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Volvariella sathei sp. nov., collected from Agharkar Research Institute Campus, Pune, is described, illustrated, and compared with morphologically closely related taxa of Volvariella and Volvopluteus. Diagnostic characters of the new species are the large white fruitbodies, relatively small basidiospores, and cylindric-clavate pleuro- and cheilocystidia. Neighbour-Joining analysis based on ITS sequences confirmed that it is distinct from its closest relative, V. nivea and other morphologically similar Volvariella species. A key to the Indian species of Volvariella and Volvopluteus is provided.

The full-potential linear augmented plane wave plus local orbital method employing the local spin-density approximation (LSDA) along with Hubbard-U corrections and spin–orbit coupling has been used to study the electronic, structural, phase transition and elastic properties of TmSb and YbSb. Under compression they undergo first-order structural transition from B1 → B2 phase. The computed phase transition pressures are 21.74 and 13.57 GPa which agrees well with their measured values ∼22.0 and 13 GPa. The structural properties viz. , equilibrium lattice constants, bulk modulus and its pressure derivative and volume collapse are also in closer agreement with their experimental data. The elastic constants and their combinations: Young's and Shear moduli, Poisson's ratio, Zener anisotropy factor, Lame's coefficients, Kleinman parameter and Debye temperature of these compounds have been computed at normal pressure. These values are in good agreement with experimental data. The LSDA + U strategy shows significant impact on the energy levels of the occupied and unoccupied 4f states in the electronic structure of both the compounds. The LSDA + U method provides better description of crystal properties of present system.

C-reactive protein (CRP) is a cyclic pentameric protein whose major binding specificity, at physiological pH, is for substances bearing exposed phosphocholine moieties. Another pentameric form of CRP, which exists at acidic pH, displays binding activity for oxidized LDL (ox-LDL). The ox-LDL-binding site in CRP, which is hidden at physiological pH, is exposed by acidic pH-induced structural changes in pentameric CRP. The aim of this study was to expose the hidden ox-LDL-binding site of CRP by site-directed mutagenesis and to generate a CRP mutant that can bind to ox-LDL without the requirement of acidic pH. Mutation of Glu(42), an amino acid that participates in intersubunit interactions in the CRP pentamer and is buried, to Gln resulted in a CRP mutant (E42Q) that showed significant binding activity for ox-LDL at physiological pH. For maximal binding to ox-LDL, E42Q CRP required a pH much less acidic than that required by wild-type CRP. At any given pH, E42Q CRP was more efficient than wild-type CRP in binding to ox-LDL. Like wild-type CRP, E42Q CRP remained pentameric at acidic pH. Also, E42Q CRP was more efficient than wild-type CRP in binding to several other deposited, conformationally altered proteins. The E42Q CRP mutant provides a tool to investigate the functions of CRP in defined animal models of inflammatory diseases including atherosclerosis because wild-type CRP requires acidic pH to bind to deposited, conformationally altered proteins, including ox-LDL, and available animal models may not have sufficient acidosis or other possible modifiers of the pentameric structure of CRP at the sites of inflammation.

A Phoma sp. was isolated and characterized as endophytic and as a pathogen of Larrea tridentata (creosote bush) growing in the desert region of southern Utah, USA. This fungus produces a unique mixture of volatile organic compounds (VOCs), including a series of sesquiterpenoids, some alcohols and several reduced naphthalene derivatives. Trans-caryophyllene, a product in the fungal VOCs, was also noted in the VOCs of this pungent plant. The gases of Phoma sp. possess antifungal properties and is markedly similar to that of a methanolic extract of the host plant. Some of the test organisms with the greatest sensitivity to the Phoma sp. VOCs were Verticillium, Ceratocystis, Cercospora and Sclerotinia while those being the least sensitive were Trichoderma, Colletotrichum and Aspergillus. We discuss the possible involvement of VOC production by the fungus and its role in the biology/ecology of the fungus/plant/environmental relationship with implications for utilization as an energy source.

A metabolite was purified from Monascus perpureus culture broth with the aim of isolating economically viable new pigment having food coloring and therapeutic potential. Following optimization of physical and nutritional parameters of submerged fermentation the pigment was extracted using hexane, and purified by thin layer chromatography (TLC), column chromatography and semi-preparative HPLC. Based on UV–vis, IR, GC–MS and NMR analyses the pigment was chemically characterized as 9-(1-hydroxyhexyl)-3-(2-hydroxypropyl)-6a-methyl-9,9a-dihydrofuro[2,3-h]isoquinoline-6,8(2H,6aH)-dione with the molecular mass of m/z 375. The pigment presents numerous similarities with two classical red pigments, rubropunctamine and monascorubramine, but differs by the presence of hydroxy alkane substitutions at C-3 and C-9 positions. Antibacterial plate assay showed its inhibitory activity against Gram-positive bacteria. These results suggest the purified pigment is a new red pigment from Monascus purpureus with potential application in food coloring and antibacterial therapy.

Thirteen strains were isolated from different habitats, belonging to two genera, namely Conidiobolus and Basidiobolus, related to saprophytic Entomophthorales. Chitin flake colonization and agar-well diffusion tests were used to screen potential extracellular chitinase-producing strains in plate assays. Preliminary screening resulted in five chitinase producers that were further studied quantitatively. Results indicated that studied isolates of this group produced chitinase at different levels in chitin-containing as well as non-chitin-containing medium. Conidioboluscoronatus was found to be the most significant chitinase producer, giving 0.261 U/ml using colloidal chitin as a carbon source, among the isolates under study. This communication also reports the chitinolytic activity of Basidiobolushaptoporus, the effect of environmental and nutritional parameters on chitinase production, and utilization of fungal biomass as a carbon source, which hitherto had not been elaborated from this genus.

PURPOSE: The mainstay imaging technique in brain tumor is Magnetic resonance imaging (MRI). However, early detection of tumor cell proliferation using MRI is limited due to inapparent disruption of normal brain architecture. Radiomics and machine learning techniques can quantitate thousands of imaging features that can depict neoplastic changes in apparently normal brain. Herein, we investigate the potential role radiomics can play in early detection of tumor cell proliferation in apparently normal MRI using a preclinically trained radiomic. METHODS: Two glioblastoma stem-like cell lines were transformed to stably express luciferase under a constitutive promoter. A stereotactic injection of tumor cells was performed to generate orthotopic mouse models (N=48). Tumor cell engraftment and in-vivo proliferation were assessed using bio-luminescence imaging (BLI) along with a weekly MRI (Bruker 7T). Images were analyzed, and ROIs were placed using 3D slicer software and radiomic features were extracted using Matlab. ROIs (0.75 mm) were placed on tumor injection sites and normal appearing contralateral brain. Radiomic features were compared for their significant alterations over time using comparative marker selection (CMS). Genomics and Histopathology of tumors were performed ex-vivo. Validation was performed in a cohort of brain cancer patients. RESULTS: Three stages of post-implantation tumor cell presence and proliferation were identified: 1. Immediate post implantation lag/engraftment phase. 2. Linear cellular proliferation phase (normal on conventional MRI). 3. Exponential cellular proliferation phase (apparent tumor on conventional MRI). Our data showed that 43% of extracted radiomic features were significantly changing (P Conclusion: Radiomic texture analysis and machine learning detects tumor cell presence and proliferation in normal-appearing brain prior to tumor development on conventional imaging. CLINICAL RELEVANCE: Radiomics and machine learning algorithms are predictive of tumor presence in seemingly normal MRIs. Early detection of tumors can allow earlier intervention, more extensive radiation planning and appropriately dose chemotherapeutic regimens.

C-reactive protein (CRP) is a phylogenetically conserved protein; in humans, it is present in the plasma and at sites of inflammation. At physiological pH, native pentameric CRP exhibits calcium-dependent binding specificity for phosphocholine. In this study, we determined the binding specificities of CRP at acidic pH, a characteristic of inflammatory sites. We investigated the binding of fluid-phase CRP to six immobilized proteins: complement factor H, oxidized low-density lipoprotein, complement C3b, IgG, amyloid β, and BSA immobilized on microtiter plates. At pH 7.0, CRP did not bind to any of these proteins, but, at pH ranging from 5.2 to 4.6, CRP bound to all six proteins. Acidic pH did not monomerize CRP but modified the pentameric structure, as determined by gel filtration, 1-anilinonaphthalene-8-sulfonic acid-binding fluorescence, and phosphocholine-binding assays. Some modifications in CRP were reversible at pH 7.0, for example, the phosphocholine-binding activity of CRP, which was reduced at acidic pH, was restored after pH neutralization. For efficient binding of acidic pH-treated CRP to immobilized proteins, it was necessary that the immobilized proteins, except factor H, were also exposed to acidic pH. Because immobilization of proteins on microtiter plates and exposure of immobilized proteins to acidic pH alter the conformation of immobilized proteins, our findings suggest that conformationally altered proteins form a CRP-ligand in acidic environment, regardless of the identity of the protein. This ligand binding specificity of CRP in its acidic pH-induced pentameric state has implications for toxic conditions involving protein misfolding in acidic environments and favors the conservation of CRP throughout evolution.

C-reactive protein (CRP) binds to Streptococcus pneumoniae through the phosphocholine groups present in the cell wall and subsequently activates the complement system to kill the pathogen. To escape the attack of complement, pneumococci recruit a complement inhibitory protein, factor H, on their surface. CRP protects mice against pneumococcal infection only when injected within 2 hours after administering pneumococci. We hypothesized that CRP is not protective when injected at later times because, by then, factor H is recruited by pneumococci. In the current study, we evaluated the protective effects of an engineered CRP molecule (E-CRP) which does not bind to factor H in fluid phase but binds to factor H-coated pneumococci. We found that E-CRP, unlike native CRP, protected mice regardless of the timing of administering E-CRP by drastically reducing bacteremia and increasing survival of mice. Next, we established another murine model of pneumococcal infection using the antibiotic clarithromycin. We found that the protective ability of 0.2 mg/dose of antibiotic was equivalent to that of 0.02 mg/dose of antibiotic combined with E-CRP; thus, there was 90% reduction in antibiotic dosing by including E-CRP in the treatment. Our data indicate that administration of E-CRP is therapeutically beneficial to reduce antibiotic dosing to decrease bacteremia and enhance survival. These results have implications for infections with antibiotic-resistant pneumococcal strains and possibly for other bacterial species that use factor H to evade the attack of complement. Additionally, the strategy to include E-CRP in the treatment using antibiotics might resolve the problem of developing antibiotic resistance.

The co-localization of plasma C-reactive protein (CRP) and atherogenic low-density lipoprotein (LDL) at the atherosclerotic lesions raises the possibility of a role of CRP in the disease process. In vitro, in its native pentameric structural form, CRP does not bind to oxidized LDL. However, in its non-native pentameric structural form, CRP is capable of binding to oxidized LDL. It has been proposed that CRP changes its structure at sites of inflammation to gain the oxidized LDL-binding activity. In vivo, native CRP is neither pro-atherosclerotic nor atheroprotective in animal models of atherosclerosis. We assumed that native CRP shows no effect because inflammatory microenvironment in the arterial wall in animal models of atherosclerosis is not appropriate and, therefore, the structure of CRP remains unchanged. Accordingly, we hypothesized that a CRP mutant, generated by site-directed mutagenesis, capable of binding to oxidized LDL without the requirement of any further structural change should show an effect on the disease. In the current study, we evaluated the impact of such a CRP mutant on the development of atherosclerosis employing LDL receptor knockout mouse model of atherosclerosis. We found that there was a two-week delay in the development of atherosclerotic lesions in the aortic root of mice treated with mutant CRP compared to mice that did not receive CRP. The development of atherosclerotic lesions in the whole aorta was also reduced; there was 30% reduction in the size of lesions in mice treated with mutant CRP. Overall, the data indicate that CRP is indeed an atheroprotective molecule.

In this paper we introduce a new species of Tamhinispora collected from Western Ghats of Maharashtra, India. This monotypic genus, belonging to Tubeufiaceae, was established based on the type species T. indica, collected from Tamhini Ghats of western India. The new species differs from the type species based on key characteristics, such as branched or Y-shaped conidial body and presence of arm-like conidial appendages. Single conidial cultures were developed and sporulation was established on MEA media following a long period of incubation. Phylogenetic analyses using ITS and LSU sequence data confirmed the position of T. srinivasanii along with T. indica in the Tamhinispora clade in Tubeufiaceae.

Assembly and guide–target interaction of an archaeal box C/D-guide sRNP was investigated under various conditions by analyzing the lead (II)-induced cleavage of the guide RNA. Guide and target RNAs derived from Haloferax volcanii pre-tRNATrp were used with recombinant Methanocaldococcus jannaschii core proteins in the reactions. Core protein L7Ae binds differentially to C/D and C′/D′ motifs of the guide RNA, and interchanging the two motifs relative to the termini of the guide RNA did not affect L7Ae binding or sRNA function. L7Ae binding to the guide RNA exposes its D′-guide sequence first followed by the D guide. These exposures are reduced when aNop5p and aFib proteins are added. The exposed guide sequences did not pair with the target sequences in the presence of L7Ae alone. The D-guide sequence could pair with the target in the presence of L7Ae and aNop5p, suggesting a role of aNop5p in target recruitment and rearrangement of sRNA structure. aFib binding further stabilizes this pairing. After box C/D-guided modification, target–guide pairing at the D-guide sequence is disrupted, suggesting that each round of methylation may require some conformational change or reassembly of the RNP. Asymmetric RNPs containing only one L7Ae at either of the two box motifs can be assembled, but a functional RNP requires L7Ae at the box C/D motif. This arrangement resembles the asymmetric eukaryal snoRNP. Observations of initial D-guide–target pairing and the functional requirement for L7Ae at the box C/D motif are consistent with our previous report of the sequential 2′-O-methylations of the target RNA.

The anti-fungal and cytotoxic activites of podophyllotoxin and seven C-4 substituted podophyllotoxin ester derivatives, viz: trans-cinnamyl, cis-cinnamyl, o-methoxy cinnamyl, dimethyl acrylyl, p-methoxy phenyl acetyl, 3,4-dimethoxy phenyl acetyl and 2,5-dimethoxy phenyl acetyl esters were evaluated on four fungi, viz: Macrophomina phaseolina, Fusarium oxysporum, Myrrothecium verrucarria and Asperigillus candidus, The podophyllotoxin derivatives were synthesised and their structures were elucidated. Quantitative structure activity relationships were developed between the activity of these compounds against the four fungi and molecular descriptors. The linear regression models developed had one to two descriptors. For all the cases the r 2 was in the range of 0.73 to 0.96, indicating good data fit and q 2 was in the range of 0.60 to 0.68, indicating that the predictive capabilities of the models were acceptable. Solvent accessible surface area (namely the partial positive solvent-accessible surface area), A log P, highest occupied molecular orbital and conformational energy were identified as important descriptors.

Different cellular and molecular effects of the alpha-2-receptoragonist Dexmedetomidine have been reported and the drug is becoming of essence in glioblastoma (GBM) care for both, awake craniotomies and in postoperative intensive care. Glioblastoma stem cells (GSCs) are an established model for preclinical GBM research. However, the effects of Dexmedetomidine on GSCs expression profile is unknown. The purpose of this research was to establish the drugs impact on the expression profiles and cellular properties of GSCs. Two independent GSC lines were exposed to Dexmedetomidine. miRNA and mRNA expression profiling by microarrays was performed using total RNA. In silico analysis were performed with ingenuity pathway analyses (IPA) and gene ontology (GO) analysis. Changes in cellular properties such as self-renewal (limiting dilution assay), differentiation (neural lineage marker analysis), proliferation (BrdU labeling), and invasion (Boyden chamber assays) were investigated upon Dexmedetomidine treatment. Dexmedetomidine treatment altered the cellular and molecular profiles of GSCs. Ingenuity pathway analyses and gene ontology analysis of differentially expressed genes in Dexmedetomidine exposed cells revealed multiple cellular functions being potentially affected. Comprehensive in vitro experiments with GSCs including differentiation, proliferation, self-renewal and migration/invasion assays show that the alpha-2-receptor-agonist can potentially impact GBM pathogenesis by significantly altering cellular behavior of GSCs. With this in silico and in vitro approach we could show how Dexmedetomidine influences GSCs on cellular and molecular level. Respecting these results offers opportunities to enhance prognosis for patients being treated for GBM by adapting the usage of this alpha-2-receptor-agonist.

A precise clinically relevant characterization of glioblastoma (GBM) heterogeneity and underlying genomic variability is urgently needed. To evaluate the true potential of radiogenomics, an emerging field for GBM research, we have tested its scope in a pre-clinical setting. First, glioblastoma stem cells (GSCs) are altered to express decreased levels of Periostin (POSTN), previously identified in a radiogenomic screen as a key pro-invasive gene. Second, orthotopic tumors arising from these GSCs, were scanned and analyzed to identify gene specific MRI texture feature (MRTF) signatures, which can simultaneously predict POSTN status in GBM patients. Doxycycline inducible short hairpin RNA mediated knockdown of POSTN in GSC lines were established. 5X10^6 cells were stereotactically implanted in nude mice frontal cortex and randomized into POSTN knockdown and control groups. Monitored tumor growth longitudinally by MRI (n = 3-4 per group). Region of interest (ROI) methods were applied to the rigid registration images based on axial T1-weighted imaging and corresponding fluid-attenuated inversion recovery sequences. After pre-processing, gray level co-occurrence matrices (GLCMs) were generated in 4 angular directions and 3 invariant measures (mean, range and angular variance) of each texture feature is calculated. A matrix of 3x20 invariant features is generated for each gray level. Mean values of normalized MRTFs from edema T1 post and T2, subgroup tumors arising from independent GSCs (p

Dexamethasone is routinely administered to manage cerebral edema in glioblastoma (GBM). Its impact on molecular mechanisms driving GBM invasion is unknown. Utilizing radiogenomic strategies, we previously introduced Periostin as key mediator of GBM associated edema and cellular invasion. We observed that multiple molecular targets of Dexamethasone are significantly upregulated and overlap with Periostin correlated genes, including CEBPB. We hypothesized that Dexamethasone, by regulating Periostin correlated genes, promotes a mesenchymal program of tumor cells including Glioblastoma Stem Cells (GSCs). A panel of GSCs were profiled for Periostin correlated genes after Dexamethasone treatment, using microarray, qPCR and Western blotting. Tests for self-renewal (limiting dilution assay), differentiation (neural lineage marker analysis), invasion (Boyden chamber) and proliferation (BrdU) were performed after Dexamethasone exposure. Three independent GSC lines were used to generate orthotopic tumors and mice were then treated with Dexamethasone (intraperitoneal). Differentially expressed genes in GSC derived tumors (total RNA from cells of human origin were isolated using human specific Anti- HLA Class I ABC antibody by FACS) upon Dexamethasone exposure were evaluated with connectivity map (cmap) to screen for specific therapeutics. Cells treated with Dexamethasone showed an increased CEBPB expression, indicating a more aggressive mesenchymal gene signature. Histopathologic validation of orthotopic tumors supported this effect of Dexamethasone. Treatment with specific, FDA approved therapeutics in combination with Dexamethasone suppressed Periostin correlated gene levels and subsequently reduced this mesenchymal shift. In this preclinical study we showed that Dexamethasone promotes a molecular mesenchymal shift by elevating CEBPB expression levels in both, in vitro and in vivo. These results warrant an evaluation of Dexamethasone for managing edema in glioblastoma patients. Neutralizing side effects of Dexamethasone by adding our shortlisted drugs may improve GBM patient care.

Our radiogenomic screen identified Periostin (POSTN) to be highly expressed in glioblastoma (GBM) with high edema/FLAIR. Subsequently, POSTN is implicated in glioblastoma invasion and recruitment of tumor associated macrophages. However, its impact on glioblastoma stem cells (GSC) biology/properties is largely unknown. Here GSC lines with high endogenous POSTN expression levels were used to generate inducible shRNA mediated loss of function (LOF) clones. GSCs with low/undetectable levels of POSTN expression were used to generate Gain of function (GOF) clones. In vitro tests were performed to assess the impact of POSTN LOF and GOF on GSC properties such as self-renewal, BrdU label retention, differentiation potential etc. We show that GSCs with POSTN LOF acquired an increased stem like state with unaltered proliferation rate. When these clones were implanted at orthotopic locations in mouse, POSTN LOF was associated with increased survival of mice. Cells of human origin were isolated from tumors using human specific HLA Class I ABC antibody by FACS. Total RNA from these sorted cells (without cell-culture) were used for expression profile analysis by HTA 2.0 array from affymetrix. Transcriptome analyses of POSTN LOF cells show inhibition of multiple mesenchymal associated networks such as CEBPB (a master regulator of mesenchymal program in GBM), PRDM1, TGFB1, FGF2, WNT3A, HIF1A etc. with concomitant activation of MYCN network, indicating a mesenchymal to proneural switch. We have generated POSTN and CEBPB dual knockdown clones and are currently investigating the molecular link between these two key mesenchymal genes in GSC context. Our results show that POSTN maintains a mesenchymal program in GSC by coopting with intracellular factors such as CEBPB. Therefore, neutralizing extracellular POSTN will have far reaching impact in terms of therapeutics where while blocking GBM invasion and it may simultaneously block a mesenchymal program in stem cell niche in GBM.

Glioblastoma (GBM) is the most aggressive human brain tumor. Although several molecular subtypes of GBM are recognized, a robust molecular prognostic marker has yet to be identified. Here, we report that the stemness regulator Sox2 is a new, clinically important target of microRNA-21 (miR-21) in GBM, with implications for prognosis. Using the MiR-21–Sox2 regulatory axis, approximately half of all GBM tumors present in the Cancer Genome Atlas (TCGA) and in-house patient databases can be mathematically classified into high miR-21/low Sox2 (Class A) or low miR-21/high Sox2 (Class B) subtypes. This classification reflects phenotypically and molecularly distinct characteristics and is not captured by existing classifications. Supporting the distinct nature of the subtypes, gene set enrichment analysis of the TCGA dataset predicted that Class A and Class B tumors were significantly involved in immune/inflammatory response and in chromosome organization and nervous system development, respectively. Patients with Class B tumors had longer overall survival than those with Class A tumors. Analysis of both databases indicated that the Class A/Class B classification is a better predictor of patient survival than currently used parameters. Further, manipulation of MiR-21–Sox2 levels in orthotopic mouse models supported the longer survival of the Class B subtype. The MiR-21–Sox2 association was also found in mouse neural stem cells and in the mouse brain at different developmental stages, suggesting a role in normal development. Therefore, this mechanism-based classification suggests the presence of two distinct populations of GBM patients with distinguishable phenotypic characteristics and clinical outcomes.SIGNIFICANCE STATEMENTMolecular profiling-based classification of glioblastoma (GBM) into four subtypes has substantially increased our understanding of the biology of the disease and has pointed to the heterogeneous nature of GBM. However, this classification is not mechanism based and its prognostic value is limited. Here, we identify a new mechanism in GBM (the miR-21–Sox2 axis) that can classify ∼50% of patients into two subtypes with distinct molecular, radiological, and pathological characteristics. Importantly, this classification can predict patient survival better than the currently used parameters. Further, analysis of the miR-21-Sox2 relationship in mouse neural stem cells and in the mouse brain at different developmental stages indicates that miR-21 and Sox2 are predominantly expressed in mutually exclusive patterns, suggesting a role in normal neural development.

International audience; This paper is a compilation of notes on 142 fungal taxa, including five new families, 20 new genera, and 100 new species, representing a wide taxonomic and geographic range. The new families, Ascocylindricaceae, Caryosporaceae and Wicklowiaceae (Ascomycota) are introduced based on their distinct lineages and unique morphology. The new Dothideomycete genera Pseudomassariosphaeria (Amniculicolaceae), Heracleicola, Neodidymella and Pseudomicrosphaeriopsis (Didymellaceae), Pseudopithomyces (Didymosphaeriaceae), Brunneoclavispora, Neolophiostoma and Sulcosporium (Halotthiaceae), Lophiohelichrysum (Lophiostomataceae), Galliicola, Populocrescentia and Vagicola (Phaeosphaeriaceae), Ascocylindrica (Ascocylindricaceae), Elongatopedicellata (Roussoellaceae), Pseudoasteromassaria (Latoruaceae) and Pseudomonodictys (Macrodiplodiopsidaceae) are introduced. The newly described species of Dothideomycetes (Ascomycota) are Pseudomassariosphaeria bromicola (Amniculicolaceae), Flammeascoma lignicola (Anteagloniaceae), Ascocylindrica marina (Ascocylindricaceae), Lembosia xyliae (Asterinaceae), Diplodia crataegicola and Diplodia galiicola (Botryosphaeriaceae), Caryospora aquatica (Caryosporaceae), Heracleicola premilcurensis and Neodidymella thailandicum (Didymellaceae), Pseudopithomyces palmicola (Didymosphaeriaceae), Floricola viticola (Floricolaceae), Brunneoclavispora bambusae, Neolophiostoma pigmentatum and Sulcosporium thailandica (Halotthiaceae), Pseudoasteromassaria fagi (Latoruaceae), Keissleriella dactylidicola (Lentitheciaceae), Lophiohelichrysum helichrysi (Lophiostomataceae), Aquasubmersa japonica (Lophiotremataceae), Pseudomonodictys tectonae (Macrodiplodiopsidaceae), Microthyrium buxicola and Tumidispora shoreae (Microthyriaceae), Alloleptosphaeria clematidis, Allophaeosphaeria cytisi, Allophaeosphaeria subcylindrospora, Dematiopleospora luzulae, Entodesmium artemisiae, Galiicola pseudophaeosphaeria, Loratospora luzulae, Nodulosphaeria senecionis, Ophiosphaerella aquaticus, Populocrescentia forlicesenensis and Vagicola vagans (Phaeosphaeriaceae), Elongatopedicellata lignicola, Roussoella magnatum and Roussoella angustior (Roussoellaceae) and Shrungabeeja longiappendiculata (Tetraploasphaeriaceae). The new combinations Pseudomassariosphaeria grandispora, Austropleospora archidendri, Pseudopithomyces chartarum, Pseudopithomyces maydicus, Pseudopithomyces sacchari, Vagicola vagans, Punctulariopsis cremeoalbida and Punctulariopsis efibulata Dothideomycetes. The new genera Dictyosporella (Annulatascaceae), and Tinhaudeus (Halosphaeriaceae) are introduced in Sordariomycetes (Ascomycota) while Dictyosporella aquatica (Annulatascaceae), Chaetosphaeria rivularia (Chaetosphaeriaceae), Beauveria gryllotalpidicola and Beauveria loeiensis (Cordycipitaceae), Seimatosporium sorbi and Seimatosporium pseudorosarum (Discosiaceae), Colletotrichum aciculare, Colletotrichum fusiforme and Colletotrichum hymenocallidicola (Glomerellaceae), Tinhaudeus formosanus (Halosphaeriaceae), Pestalotiopsis subshorea and Pestalotiopsis dracaenea (Pestalotiopsiceae), Phaeoacremonium tectonae (Togniniaceae), Cytospora parasitica and Cytospora tanaitica (Valsaceae), Annulohypoxylon palmicola, Biscogniauxia effusae and Nemania fusoideis (Xylariaceae) are introduced as novel species to order Sordariomycetes. The newly described species of Eurotiomycetes are Mycocalicium hyaloparvicellulum (Mycocaliciaceae). Acarospora septentrionalis and Acarospora castaneocarpa (Acarosporaceae), Chapsa multicarpa and Fissurina carassensis (Graphidaceae), Sticta fuscotomentosa and Sticta subfilicinella (Lobariaceae) are newly introduced in class Lecanoromycetes. In class Pezizomycetes, Helvella pseudolacunosa and Helvella rugosa (Helvellaceae) are introduced as new species. The new families, Dendrominiaceae and Neoantrodiellaceae (Basidiomycota) are introduced together with a new genus Neoantrodiella (Neoantrodiellaceae), here based on both morphology coupled with molecular data. In the class Agaricomycetes, Agaricus pseudolangei, Agaricus haematinus, Agaricus atrodiscus and Agaricus exilissimus (Agaricaceae), Amanita melleialba, Amanita pseudosychnopyramis and Amanita subparvipantherina (Amanitaceae), Entoloma calabrum, Cora barbulata, Dictyonema gomezianum and Inocybe granulosa (Inocybaceae), Xerocomellus sarnarii (Boletaceae), Cantharellus eucalyptorum, Cantharellus nigrescens, Cantharellus tricolor and Cantharellus variabilicolor (Cantharellaceae), Cortinarius alboamarescens, Cortinarius brunneoalbus, Cortinarius ochroamarus, Cortinarius putorius and Cortinarius seidlii (Cortinariaceae), Hymenochaete micropora and Hymenochaete subporioides (Hymenochaetaceae), Xylodon ramicida (Schizoporaceae), Colospora andalasii (Polyporaceae), Russula guangxiensis and Russula hakkae (Russulaceae), Tremella dirinariae, Tremella graphidis and Tremella pyrenulae (Tremellaceae) are introduced. Four new combinations Neoantrodiella gypsea, Neoantrodiella thujae (Neoantrodiellaceae), Punctulariopsis cremeoalbida, Punctulariopsis efibulata (Punctulariaceae) are also introduced here for the division Basidiomycota. Furthermore Absidia caatinguensis, Absidia koreana and Gongronella koreana (Cunninghamellaceae), Mortierella pisiformis and Mortierella formosana (Mortierellaceae) are newly introduced in the Zygomycota, while Neocallimastix cameroonii and Piromyces irregularis (Neocallimastigaceae) are introduced in the Neocallimastigomycota. Reference specimens or changes in classification and notes are provided for Alternaria ethzedia, Cucurbitaria ephedricola, Austropleospora, Austropleospora archidendri, Byssosphaeria rhodomphala, Lophiostoma caulium, Pseudopithomyces maydicus, Massariosphaeria, Neomassariosphaeria and Pestalotiopsis montellica.

Reactions of stable N-(o-hydroxyarylacyl)benzotriazoles 4a–c / 6a–c with various aldehydes in the presence of base afforded 1,3-benzodioxin-4-ones 1a–d and naphtho-1,3-dioxinones 7a–c. Reaction with isocyanates under similar conditions afforded high yields of benzoxazine-2,4diones 2a–c and naphthoxazine-1,3-diones 9a–b.

Present study aims to enhance the efficacy of liposomes as an adjuvant by steric protection and strengthen the path of vaccine research. PEG grafted liposomes carrying epitopes on their surface showed enhanced adjuvanticity than liposomes carrying epitopes for elicitation and prolongation of immune response to an antigenic epitope of gp41, a transmembrane protein of HIV-1. The multiples of epitope were incorporated onto the surface of liposomes by conjugating them with phosphatidylethanolamine that was used in the formulation of liposomes at an optimized ratio. Furthermore, the liposomes carrying epitopes on their surface were sterically protected by shielding with methoxy-poly(ethylene glycol), mass 20 kDa. Methoxy-poly(ethylene glycol) was activated to its electrophilic N-succinimide carbonate derivative, methoxy-poly(ethylene glycol)-N-succinimide carbonate, that formed a urethane linkage with the amino group of phosphatidylethanolamine. The epitope was covalently coupled to phosphatidylethanolamine through an amide bond between the -COOH group of the epitope and -NH2 group of phosphatidylethanolamine under the catalysis of 1-ethyl-3-(3-dimethylaminopropy-1)-carbodiimide. PEG grafted epitopes carrying liposomes showed about two times higher immune response and prolonged persistence of antibodies than that of liposomes carrying epitopes without PEG moieties.

Recent reviews of the Software Quality Assurance (SQA) literature expose that in most organizations, systems development is characterized by recurrent problems, such as poor system quality. The conclusions of these reviews is that SQA efforts have failed due primarily to their failure to adequately address the linkages between the organizational and process aspects of quality assurance in software development projects. As an initial step in identifying and understanding the linkages between the organizational and process aspects of SQA, the authors conducted an exploratory study to elicit a set of issues critical to the assurance of delivering reliable software.

Three different methods are reported for the preparation of 4-azacyclo-2-enones 1, two of which allow the preparation of the compounds in optically active form. In addition, a facile route to 4-aza-5,5-dimethylcyclopent-2-enones 2 is disclosed.

Imaging is the modality of choice for noninvasive characterization of biological tissue and organ systems; imaging serves as early diagnostic tool for most disease processes and is rapidly evolving, thus transforming the way we diagnose and follow patients over time. A vast number of cancer imaging characteristics have been correlated to underlying genomics; however, none have established causality. Therefore, our objectives were to test if there is a causal relationship between imaging and genomic information; and to develop a clinically relevant radiomic pipeline for glioblastoma molecular characterization. Functional validation was performed using a prototypic in vivo RNA-interference-based orthotopic xenograft mouse model. The automated pipeline collects 4800 MRI-derived texture features per tumor. Using univariate feature selection and boosted tree predictive modeling, a patient-specific genomic probability map was derived and patient survival predicted (The Cancer Genome Atlas/MD Anderson data sets). Data demonstrated a significant xenograft to human association (area under the curve [AUC] 84%, P

To report treatment setup data from an emerging technique using near-simultaneous computed tomography (CT) image-guided stereotactic radiotherapy for the treatment of spinal and paraspinal tumors.A targeting system that integrates a CT-on-rails scanner with a linear accelerator (LINAC) was evaluated in the lead-in portion of a Phase I/II protocol for treating patients with paraspinal metastases. Patients were immobilized in supine position by a moldable body cushion vacuum wrapped with a plastic fixation sheet. Planning CT and immediately repeated CT were performed on the LINAC/CT-on-rails unit to assess respiratory-related vertebral body motion. Coplanar intensity-modulated radiotherapy (IMRT) using 7-9 beams was used to deliver 30 Gy in five fractions to the target volume, while limiting the spinal cord dose to10 Gy. Pretreatment CT scans were fused with the planning CT scans to determine the correct target isocenter by accounting for any translational and roll (axial) rotational discrepancies from the planning CT. (Corrections caused by yaw and pitch rotations have not yet been implemented.) The reproducibility of the treatment isocenter as compared with the planned isocenter was measured with digitally reconstructed radiographs (DRRs), portal film imaging, and immediate post-treatment verification CT scans. Phantom measurements were taken for dose verification for each IMRT plan.Based on a total of 36 CT scans (3 for planning, 3 for respiration study, 15 pretreatment, and 15 post-treatment) from 3 patients, no respiration-associated vertebral body motion was seen. A comparison of the corrected daily anterior-posterior (AP) and lateral (LAT) digital portal images with the planning AP and LAT DRRs confirmed that the isocenter setup accuracy for the 15 treatments was within 1 mm of the planning isocenter. The results from the immediate post-treatment CT scans reconfirmed the findings from the portal images and verified the absence of spinal movement during the treatment. The ion-chamber measurement for the high-dose region was within 2% of the planning dose for three patient treatment plans. Film dose measurement in an IMRT quality assurance phantom demonstrated good agreement from 90% to 30% isodose lines between the planned and measured results.Preliminary experience suggests that the near-simultaneous CT image-guided verification technique can be used as a new platform technology for extracranial applications of stereotactic radiotherapy and radiosurgery to spinal and paraspinal tumors.

OBJECTIVE We compared the ability of proton magnetic resonance spectroscopic imaging (1H-MRSI) measures with that of standard clinicopathological measures to predict length of survival in patients with supratentorial gliomas. METHODS We developed two sets of leave-one-out logistic regression models based on either 1) intratumoral 1H-MRSI features, including maximum values of a) choline and b) lactate-lipid, c) number of 1H-MRSI voxels with low N-acetyl group values, and d) number of 1H-MRSI voxels with high lactate-lipid values, all (a–d) of which were normalized to creatine in normal-appearing brain, or 2) standard clinicopathological features, including a) tumor histopathological grade, b) patient age, c) performance of surgical debulking, and d) tumor diagnosis (i.e., oligodendroglioma, astrocytoma). We assessed the accuracy of these two models in predicting patient survival for 6, 12, 24, and 48 months by performing receiver operating characteristic curve analysis. Cox proportional hazards analysis was performed to assess the extent to which patient survival could be explained by the above predictors. We then performed a series of leave-one-out linear multiple regression analyses to determine how well patient survival could be predicted in a continuous fashion. RESULTS The results of using the models based on 1H-MRSI and clinicopathological features were equally good, accounting for 81 and 64% of the variability (r2) in patients' actual survival durations. All features except number of 1H-MRSI voxels with lactate-lipid/creatine values of at least 1 were significant predictors of survival in the 1H-MRSI model. Two features (tumor grade and debulking) were found to be significant predictors in the clinicopathological model. Survival as a continuous variable was predicted accurately on the basis of the 1H-MRSI data (r = 0.77, P < 0.001; median prediction error, 1.7 mo). CONCLUSION Our results suggest that appropriate analysis of 1H-MRSI data can predict survival in patients with supratentorial gliomas at least as accurately as data derived from more invasive clinicopathological features.

Archaeal RNA splicing involves at least two protein enzymes, a specific endonuclease and a specific ligase. The endonuclease recognizes and cleaves within a characteristic bulge-helix-bulge (BHB) structure formed by pairing of the regions near the two exon–intron junctions, producing 2‘,3′-cyclic phosphate and 5′-hydroxyl termini. The ligase joins the exons and converts the cyclic phosphate into junction phosphate. The ligated product contains a seven-base hairpin loop, in which the splice junction is in between the two 3′ terminal residues of the loop. Archaeal splicing endonucleases are also involved in rRNA processing, cutting within the BHB structures formed by pairing of the 5′ and 3′ flanking regions of the rRNAs. Large free introns derived from pre-rRNAs have been observed as stable and abundant circular RNAs in certain Crenarchaeota, a kingdom in the domain Archaea. In the present study, we show that the cells of Haloferax volcanii, a Euryarchaeote, contain circular RNAs formed by 3′,5′-phosphodiester linkage between the two termini of the introns derived from their pre-tRNAs. H. volcanii ligase, in vitro, can also circularize both endonuclease-cleaved introns, and non-endonuclease-produced substrates. Exon joining and intron circularization are mechanistically similar ligation reactions that can occur independently. The size of the ligated hairpin loop and position of the splice junction within this loop can be changed in in vitro ligation reactions. Overall, archaeal RNA splicing seems to involve two sets of two symmetric transesterification reactions each.

A novel furan based scaffold 5-hydroxymethylfurfural has been identified for the generation of combinatorial libraries using template directed approach on solid phase. This scaffold has been utilized to afford furan-based bi-heterocyclic structures with extensive chemical diversity using cycloaddition, multicomponent and cyclization reactions.

To identify the molecular profiles of cell death as defined by necrosis volumes at magnetic resonance (MR) imaging and uncover sex-specific molecular signatures potentially driving oncogenesis and cell death in glioblastoma (GBM).This retrospective study was HIPAA compliant and had institutional review board approval, with waiver of the need to obtain informed consent. The molecular profiles for 99 patients (30 female patients, 69 male patients) were identified from the Cancer Genome Atlas, and quantitative MR imaging data were obtained from the Cancer Imaging Archive. Volumes of necrosis at MR imaging were extracted. Differential gene expression profiles were obtained in those patients (including male and female patients separately) with high versus low MR imaging volumes of tumor necrosis. Ingenuity Pathway Analysis was used for messenger RNA-microRNA interaction analysis. A histopathologic data set (n = 368; 144 female patients, 224 male patients) was used to validate the MR imaging findings by assessing the amount of cell death. A connectivity map was used to identify therapeutic agents potentially targeting sex-specific cell death in GBM.Female patients showed significantly lower volumes of necrosis at MR imaging than male patients (6821 vs 11 050 mm(3), P = .03). Female patients, unlike male patients, with high volumes of necrosis at imaging had significantly shorter survival (6.5 vs 14.5 months, P = .01). Transcription factor analysis suggested that cell death in female patients with GBM is associated with MYC, while that in male patients is associated with TP53 activity. Additionally, a group of therapeutic agents that can potentially be tested to target cell death in a sex-specific manner was identified.The results of this study suggest that cell death in GBM may be driven by sex-specific molecular pathways.

Cellular heterogeneity is one of the key contributors to poor clinical outcome in glioblastoma (GBM). In this issue Lathia and colleagues1 report on the development of a novel mouse model where the GFAP-tva mouse2 harbors a Sox2-EGFP reporter3 to enable direct visualization of intratumoral cellular heterogeneity in a RCAS-PDGFβ-driven4 GBM model. Cellular heterogeneity is associated with one of the salient features of glioblastoma stem cells (GSCs). Multiple studies have elucidated that GSCs are a unique subpopulation of cells within GBM and are responsible for hierarchical cellular organization.5 Additionally, GSCs may potentially orchestrate multiple other aspects of GBM biology, including resistance to therapy, tumor recurrence, and cellular invasion, thus contributing to dismal clinical outcomes despite aggressive therapy.6 The characterizations of GSCs isolated/enriched from surgical human GBM specimens have lent support to the cancer stem cell hypothesis and have opened avenues for understanding the complex dynamics of cancer progression, recurrence, and resistance to therapy. Thus far, marker-based enrichment of GSCs has remained controversial due to the potential dissociation from their ‘stem-cell state’ once removed from their native tumor niche (e.g. perivascular7).

Glioblastoma multiforme (GBM) is characterized by a pathogenic vasculature that drives aggressive local invasion. Recent work suggests that GBM cells recruit bone marrow–derived progenitor cells (BMDC) to facilitate recurrence after radiotherapy, but how this may be achieved is unclear. In this study, we established the spatiotemporal and regional contributions of perivascular BMDCs (pBMDC) to GBM development. We found an increased recruitment of BMDCs to GBM in response to tumor growth and following radiotherapy. However, in this study, BMDCs did not differentiate into endothelial cells directly but rather provided a perivascular support role. The pBMDCs were shown to associate with tumor vasculature in a highly region-dependent manner, with central vasculature requiring minimal pBMDC support. Region-dependent association of pBMDC was regulated by VEGF. In the absence of VEGF, following radiotherapy or antiangiogenic therapy, we documented an increase in Ang2 that regulated recruitment of pBMDCs to maintain the vulnerable central vasculature. Together, our results strongly suggested that targeting pBMDC influx along with radiation or antiangiogenic therapy would be critical to prevent vascular recurrence of GBM. Cancer Res; 74(14); 3727–39. ©2014 AACR.

To compare contrast material-enhanced T1-weighted and fluid-attenuated inversion-recovery (FLAIR) magnetic resonance (MR) images in depicting leptomeningeal metastases.Malignant lesions detected at cytologic examination of cerebrospinal fluid in 70 patients were reviewed. There were 58 studies in which both FLAIR and contrast-enhanced T1-weighted spin-echo MR images were available. A senior neuroradiologist reviewed the images from each sequence individually and separately for signs of leptomeningeal metastases and assigned a diagnostic rating of positive, indeterminate, or negative.Leptomeningeal metastases were depicted in 38 cases on contrast-enhanced T1-weighted spin-echo images and in 20 cases on FLAIR images. In three cases, leptomeningeal metastases were detected by using only FLAIR images. In 20 cases, leptomeningeal metastases were detected by using only contrast-enhanced T1-weighted spin-echo images. FLAIR imaging has a sensitivity of 34% for cytologically proved leptomeningeal metastases. Gadolinium-enhanced MR imaging has a sensitivity of 66%.Used alone, contrast-enhanced T1-weighted images are better than FLAIR images for detecting leptomeningeal metastases. This is particularly true for cases in which leptomeningeal metastases manifest primarily or solely as cranial nerve involvement.

We have used NMR and CD spectroscopy to study the conformations of modified oligonucleotides (locked nucleic acid, LNA) containing a conformationally restricted nucleotide (T(L)) with a 2'-O,4'-C-methylene bridge. We have investigated two LNA:RNA duplexes, d(CTGAT(L)ATGC):r(GCAUAUCAG) and d(CT(L)GAT(L)AT(L)GC):r(GCAUAUCAG), along with the unmodified DNA:RNA reference duplex. Increases in the melting temperatures of +9.6 degrees C and +8.1 degrees C per modification relative to the unmodified duplex were observed for these two LNA:RNA sequences. The three duplexes all adopt right-handed helix conformations and form normal Watson-Crick base pairs with all the bases in the anti conformation. Sugar conformations were determined from measurements of scalar coupling constants in the sugar rings and distance information derived from 1H-1H NOE measurements; all the sugars in the RNA strands of the three duplexes adopt an N-type conformation (A-type structure), whereas the sugars in the DNA strands change from an equilibrium between S- and N-type conformations in the unmodified duplex towards more of the N-type conformation when modified nucleotides are introduced. The presence of three modified T(L) nucleotides induces drastic conformational shifts of the remaining unmodified nucleotides of the DNA strand, changing all the sugar conformations except those of the terminal sugars to the N type. The CD spectra of the three duplexes confirm the structural changes described above. On the basis of the results reported herein, we suggest that the observed conformational changes can be used to tune LNA:RNA duplexes into substrates for RNase H: Partly modified LNA:RNA duplexes may adopt a duplex structure between the standard A and B types, thereby making the RNA strand amenable to RNase H-mediated degradation.

The authors evaluated the RNA binding of all eight possible stereoisomers of LNA. Synthesis of \"a-l-xylo-LNA\" has been accomplished and the remaining four stereoisomers, all enantiomers of the four synthesized diastereoisomers, are indirectly evaluated by hybridization studies. The remarkable binding affinities and specificities obtained for LNA and a-l-LNA, both in a fully and in a partly modified context, establish these mols. as unique nucleic acid mimics. A strong impetus for this research has been, and continues to be, the therapeutic promises of the antisense strategy. [on SciFinder (R)]

We have used two-dimensional (1)H NMR spectroscopy at 750 MHz to determine a high-resolution solution structure of an oligonucleotide containing restricted nucleotides with a 2'-O, 4'-C-methylene bridge (LNA) hybridized to the complementary DNA strand. The LNA:DNA duplex examined contained four thymidine LNA modifications (T(L), d(C1T(L)2G3C4T(L)5T(L)6C7T(L)8G9C10):d( G11C12A13G14A15A16G17C 18A19G20). A total relaxation matrix approach was used to obtain interproton distance bounds from NOESY cross-peak intensities. These distance bounds were used as restraints in molecular dynamics (rMD) calculations. Forty final structures were generated for the duplex from A-form and B-form DNA starting structures. The root-mean-square deviation (RMSD) of the coordinates for the 40 structures of the complex was 0.6 A. The sugar puckerings are averaged values of a dynamic interchange between N- and S-type conformation except in case of the locked nucleotides that were found to be fixed in the C3'-endo conformation. Among the other nucleotides in the modified strand, the furanose ring of C7 and G9 is predominantly in the N-type conformation whereas that of G3 is in a mixed conformation. The furanose rings of the nucleotides in the unmodified complementary strand are almost exclusively in the S-type conformation. Due to these different conformations of the sugars in the two strands, there is a structural strain between the A-type modified strand and the B-type unmodified complementary strand. This strain is relaxed by decreasing the value of rise and compensating with tip, buckle, and propeller twist. The values of twist vary along the strand but for a majority of the base pairs a value even lower than that of A-DNA is observed. The average twist over the sequence is 32+/-1 degrees. On the basis of the structure, we conclude that the high stability of LNA:DNA duplexes is caused by a local change of the phosphate backbone geometry that favors a higher degree of stacking.

We have used 2D NMR spectroscopy to study the sugar conformations of oligonucleotides containing a conformationally restricted nucleotide (LNA) with a 2'-O, 4'-C-methylene bridge. We have investigated a modified 9-mer single stranded oligonucleotide as well as three 9- and 10-mer modified oligonucleotides hybridized to unmodified DNA. The single-stranded LNA contained three modifications whereas the duplexes contained one, three and four modifications, respectively. The LNA:DNA duplexes have normal Watson-Crick base-pairing with all the nucleotides in anti-conformation. By use of selective DQF-COSY spectra we determined the ratio between the N-type (C3'-endo) and S-type (C2'-endo) sugar conformations of the nucleotides. In contrast to the corresponding single-stranded DNA (ssDNA), we found that the sugar conformations of the single-stranded LNA oligonucleotide (ssLNA) cannot be described by a major S-type conformer of all the nucleotides. The nucleotides flanking an LNA nucleotide have sugar conformations with a significant population of the N-type conformer. Similarly, the sugar conformations of the nucleotides in the LNA:DNA duplexes flanking a modification were also shown to have significant contributions from the N-type conformation. In all cases, the sugar conformations of the nucleotides in the complementary DNA strand in the duplex remain in the S-type conformation. We found that the locked conformation of the LNA nucleotides both in ssLNA and in the duplexes organize the phosphate backbone in such a way as to introduce higher population of the N-type conformation. These conformational changes are associated with an improved stacking of the nucleobases. Based on the results reported herein, we propose that the exceptional stability of the LNA modified duplexes is caused by a quenching of concerted local backbone motions (preorganization) by the LNA nucleotides in ssLNA so as to decrease the entropy loss on duplex formation combined with a more efficient stacking of the nucleobases.

LNA (Locked Nucleic Acids) is a novel oligonucleotide analogue containing a conformationally restricted nucleotide with a 2'-O, 4'-C-methylene bridge that induces unprecedented thermal affinities when mixed with complementary single stranded DNA and RNA. We have used two-dimensional 1H NMR spectroscopy obtained at 750 and 500 MHz to determine a high resolution solution structure of an LNA oligonucleotide hybridized to the complementary DNA strand. The determination of the structure was based on a complete relaxation matrix analysis of the NOESY cross peaks followed by restrained molecular dynamics calculations. Forty final structures were generated for the duplex from A-type and B-type dsDNA starting structures. The root-mean-square deviation (RMSD) of the coordinates for the forty structures of the complex was 0.32A. The structures were analysed by use of calculated helix parameters. This showed that the values for rise and buckle in the LNA duplex is markedly different from canonical B-DNA at the modification site. A value of twist similar to A-DNA is also observed at the modification site. The overall length of the helix which is 27.3 A. The average twist over the sequence are 35.9 degrees +/- 0.3 degrees. Consequently, the modification does not cause the helix to unwind. The bis-intercalation of the thiazole orange dye TOTO to the LNA duplex was also investigated by 1H NMR spectroscopy to sense the structural change from the unmodified oligonucleotide. We observed that the bis-intercalation of TOTO is much less favourable in the 5'-CT(L)AG-3' site than in the unmodified 5'-CTAG-3' site. This was related to the change in the base stacking of the LNA duplex compared to the unmodified duplex.

LNA (Locked Nucleic Acids), consisting of 2′- O ,4′- C -methylene bicyclonucleoside monomers, is efficiently synthesized and its nucleic acid recognition potential evaluated for six different nucleobases, namely adenine, cytosine, guanine, 5-methylcytosine, thymine and uracil. Unprecedented increases (+3 to +8 °C per modification) in the thermal stability of duplexes towards both DNA and RNA were obtained when evaluating mixed sequences of partly or fully modified LNA. Studies of mis-matched sequences show that LNA obey the Watson-Crick base pairing rules with generally improved selectivities compared to the corresponding unmodified reference strands.

Aflatoxin B, (AFB1) is a clastogen that causes cellular damage by covalent modification of nucleic acids. In this investigation, male rats were injected i.p. with AFB1 (8 mg/kg b.w.) in DMSO and the same dose of AFB1 was also administered intratracheally (i.t.) to the animals separately. The animals were killed after 26 h of the carcinogen treatment, femur bone was removed, and bone marrow cells were isolated and stained with Mayer's hematoxylin and eosin. Micronuclei (Mn) were scored by using light microscopy. Bronchoalveolar lavage (BAL) was prepared from rats administered AFB1 i.t. A part of BAL was fixed with 70% ethanol, stained with the fluorochrome DAPI, and analysed for cell cycle variations; the other part of the lavage was used for making slides to record Mn with a fluorescent microscope. A significantly greater proportion of lung cells were found to enter cell cycle with extended S-phase due to AFB1 treatment. Mn were induced in polychromatic erythrocytes (PCE) as compared to normochromatic erythrocytes (NCE) in the bone marrow of AFB1-treated rats, where there was nearly a three-fold increase in the number of Mn of bone marrow cells. The administration of AFB1 resulted in a two-fold rise in the Mn in the lung cells. The effect of BSO, DEM, and PB, the modulators of AFB1 metabolism, was studied on AFB1-induced Mn formation. A significant increase in the Mn score in PCEs of BSO- and DEM-treated rats was noted, while a slight reduction in the Mn score was noted in the case of PB-treated rats. The administration of the methanol extract of the leaves of Piper argyrophyllum (taken up in DMSO) to rats for a week exhibited normalising effect on AFB1-induced Mn in bone marrow cells. These observations record the induction of Mn in lung cells due to AFB1 for the first time. We propose the utility of AFB1-induced Mn as a model for screening plant extracts as inhibitors of genotoxicity. Prevention of genotoxic changes described above by phytochemicals is being pursued in our Laboratories.