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Ratoon stunting disease (RSD), caused by the bacterium Leifsonia xyli subsp. xyli, is arguably one of the most devastating diseases of sugarcane. Four diagnostic techniques were compared for 100 fields of sugarcane (Saccharum interspecific hybrids) of unknown infection status. These were quantitative polymerase chain reaction on pooled leaf sheath biopsies (LSB-qPCR), conventional PCR on the same templates (LSB-PCR), evaporative-binding enzyme immunoassay (EB-EIA) coupled with phase contrast microscopy (PCM) on expressed xylem sap from the same fields, and conventional PCR on the same xylem sap samples. LSB-qPCR and LSB-PCR detected the causal agent in 27 and 18 fields, respectively, whereas, from samples of expressed xylem sap from the same fields, conventional PCR identified 12 infections and EB-EIA/PCM detected L. xyli subsp. xyli in 3 fields. The sensitivities of qPCR and PCR were approximately 103 and 104 CFU ml−1, respectively, determined from plate counts of a dilution series. Tests were conducted on a further 139 LSB samples from across the Australian industry, with qPCR and PCR diagnosing RSD in 31 and 25 fields, respectively. Using qPCR and PCR on LSB samples, RSD was diagnosed in a range of cultivars throughout the year, and qPCR and PCR could detect L. xyli subsp. xyli in sugarcane ranging from 3 months to greater than 1 year old.

Ethnopharmacological relevance Australian Aboriginal people used crushed leaves of Geijera parviflora Lindl. both internally and externally for pain relief, including for toothache ( Cribb and Cribb, 1981 ). This study tested the hypothesis that this traditional use might be at least in part explained by the presence of compounds with anti-inflammatory activity. Materials and methods A crude extract (95% EtOH) was prepared from powdered dried leaves. From the CH 3 Cl fraction of this extract compounds were isolated by bioassay-guided fractionation and tested for: (1) cytotoxicity in RAW 264.7 murine leukemic monocyte–macrophages, (2) prostaglandin E 2 (PGE 2 ) inhibitory activity in 3T3 Swiss albino mouse embryonic fibroblast cells, as well as (3) nitric oxide (NO) and (4) tumour necrosis factor alpha (TNFα) inhibitory activity in lipopolysaccharide (LPS)-stimulated RAW 264.7 cells. Isolated compounds were also tested for (5) antibacterial activity against a panel of Gram-positive ( Staphylococcus aureus ATCC 29213 and ATCC 25923, Staphylococcu s epidermidis ATCC 35984, biofilm-forming) and Gram-negative ( Escherichia coli ATCC 25922, Pseudomonas aeruginosa ATCC 27853) strains by broth microdilution. Results Eleven compounds were isolated, including one new flavone and one new natural product, with a further four compounds reported from this species for the first time. Some of the compounds showed good anti-inflammatory activity in vitro . In particular, flindersine ( 1 ) and N-(acetoxymethyl) flindersine ( 3 ) inhibited PGE 2 release with IC 50 values of 5.0 μM and 4.9 μM, respectively, without any significant cytotoxicity. Several other compounds showed moderate inhibition of NO ( 5 , 6 , 7 ) and TNF-α ( 6) , with IC 50 in the low micromolar range; however much of this apparent activity could be accounted for by the cytotoxicity of these compounds. None of the compounds showed anti-bacterial activity. Conclusions The inhibition of PGE 2 , an important mediator of inflammation and pain, by flindersine and a derivative thereof, along with the moderate anti-inflammatory activity shown by several other compounds isolated from Geijera parviflora leaf extract, support the traditional use of this plant for pain relief by Australian Aboriginal people.

In an effort to identify new anti-inflammatory and antibacterial agents with potential application in wound healing, five new dibenzofurans, 1,3,7,9-tetrahydroxy-2,8- dimethyl-4,6-di(2-methylbutanoyl)dibenzofuran (1), 1,3,7,9- tetrahydroxy-2,8-dimethyl-4-(2-methylbutanoyl)-6-(2- methylpropionyl)dibenzofuran (2), 1,3,7,9-tetrahydroxy-2,8- dimethyl-4,6-di(2-methylpropionyl)dibenzofuran (3), 1,3,7,9- tetrahydroxy-4,6-dimethyl-2-(2-methylbutanoyl)-8-(2- methylpropionyl)dibenzofuran (4), and 1,3,7,9-tetrahydroxy- 4,6-dimethyl-2,8-di(2-methylpropionyl)dibenzofuran (5), were isolated from the leaves of Pilidiostigma glabrum together with one previously described dibenzofuran. Structure elucidation was achieved by way of spectroscopic measurements including 2D-NMR spectroscopy. Compounds with 2,8-acyl substitutions had potent antibacterial activity against several Gram-positive strains (MIC in the low micromolar range), while compounds with 4,6- acyl substitutions were less active. All compounds except 3 inhibited the synthesis of nitric oxide in RAW264 macrophages with IC50 values in the low micromolar range. Compounds with 2,8-acyl substitutions also inhibited the synthesis of PGE2 in 3T3 cells, whereas 4,6-acyl-substituted compounds were inactive. None of the compounds inhibited the synthesis of TNF-α in RAW264 cells. The compounds showed variable but modest antioxidant activity in the oxygen radical absorbance capacity assay. These findings highlight that much of the Australian flora remains unexplored and may yet yield many new compounds of interest. Initial clues are provided on structure/activity relationships for this class of bioactives, which may enable the design and synthesis of compounds with higher activity and/or selectivity. Refereed/Peer-reviewed

Pilidiostigmin, a novel dimeric acylphloroglucinol derivative, was isolated from the leaves of the Australian plant species Pilidiostigma glabrum (Myrtaceae). Pilidiostigmin exists in the plant in the form of a sodium salt, which is rare in natural products. The elucidation of the structure and relative configuration was achieved by spectroscopic measurements with special emphasis on 1D and 2D NMR techniques. Pilidiostigmin was found to display biological activity; it significantly and dose-dependently inhibited the synthesis of nitric oxide in LPS-stimulated RAW 264.7 macrophages at low micromolar concentrations. (C) 2013 Elsevier Ltd. All rights reserved.

Many viruses have evolved mechanisms to antagonize the interferon (IFN) system, targeting all the major components involved in receptor binding and signaling. Although a number of these vital proteins are homologous to cellular proteins involved in IFN downregulation (e.g., viral IFN regulatory factors [vIRFs]), many share little resemblance to known proteins. To determine the IFN-blocking properties of these proteins, functional assays are required. Here, we present a new and rapid functional screening method, based on the 2fTGH cell line, which is able to determine viral gene products that inhibit the IFN-alpha/Jak-Stat signaling pathway. Expression cloning of viral IFN-blocking genes into 2fTGH and consequent selection with IFN-alpha and 6-thioguanine result in the outgrowth of cells that are no longer responsive to IFN-alpha. We also demonstrate that selection occurs if members of the Jak-Stat signaling pathway are lost. To show the utility of our system, we have used a known suppressor of IFN signaling, the human papillomavirus (HPV) E7 gene. Expression of E7 causes the loss of ability of 2fTGH cells to respond to IFN-alpha treatment because of a functional disruption of the signaling pathway. This approach offers a new strategy for identifying novel viral genes or new functions of already described viral genes that have a role in IFN-alpha signaling inhibition.