Therapeutic effects of beta-thujaplicin eardrops on canine Malassezia-related otitis externa

An eardrop solution of beta-thujaplicin was examined for therapeutic effects on canine Malassezia-related otitis externa. Half to one ml of beta-thujaplicin solution of 100 microg/ml including DMSO 2% was injected everyday into both external ear canals of 31 cases for test-of-cure agreement. Fifteen score phases were established from the symptoms and cerumen smear biopsy findings, and score changes were recorded at least once a week. The means of the second and the third inspection day scores decreased significantly more than the previous value of each. In addition, the numbers of yeast-like organisms clearly decreased. These results suggest that beta-thujaplicin eardrops are effective for Malassezia-related otitis externa in dogs.     

Therapeutic potential of canine bone marrow stromal cells (BMSCs) in the carbon tetrachloride (CCl4) induced chronic liver dysfunction mouse model

Regenerative medicine using bone marrow cells is an attractive therapy for the cure of patients with severe liver disease. Here, we show the therapeutic potential of canine bone marrow stromal cells (BMSCs) in mouse models of CCl(4)-induced chronic liver dysfunction. We used two different models for xenotransplantation, nude mice and cyclosporine A (CSA) immunosuppressed mice. Serum parameters from a standard liver panel were not improved following transplantation. However, fibrotic liver lesions with severe inflammation were decreased in CCl(4)-treated CSA mice following BMSC transplantation. Effective migration of transplanted canine BMSCs was limited to persistently injured liver in CCl(4)-treated CSA mice, where they may be effective in resolving inflammatory fibrotic lesions. These results suggest that canine BMSCs are an effective cell source for liver regeneration.     

Pruning methods to restore Castanea sativa stands attacked by Dryocosmus kuriphilus

Dryocosmus kuriphilus Yasumatsu, an invasive gall wasp originating from Asia, is the latest serious pest attacking Castanea sativa Mill. in Europe; the negative effects on fruit and wood production are considerable. The most effective defense strategy is biological control, but parasitoid effectiveness requires at least 6–10 years to reduce damage to acceptable levels. In Italy, prolonged productivity losses could adversely affect human activity in chestnut orchard stands, resulting in negative socio-economic and hydrogeologic consequences. Thus, it is necessary to quickly reduce the impacts of the infestation to avoid degradation of chestnut stands during the time delay required for parasitoid efficiency. We examined green pruning as a means to reduce negative impacts of gall wasps and stimulate healthy shoots in a young chestnut coppice in Central Italy. Two different pruning cuts (Long- and Short-cut) on growing shoots and four timings of application were tested. The development and phytosanitary status of nodes was observed over 2 years on pruned and control sprouts. Pruning was effective in reducing gall wasp damage though responses varied among pruning techniques and application times. Long-cut pruning made in spring during the full growth phase induced a higher number of new and better-developed shoots. Pruning during the end of the egg-laying period of the gall wasps promoted development of only healthy new shoots. Our results indicate that is possible, by choosing the best combination of time and technique, to identify a pruning method useful to maintain growth and development of C. sativa stands threatened by D. kuriphilus.

     

Effect of inter-annual climate variability on evapotranspiration and canopy CO2 exchange of a tropical rainforest in Peninsular Malaysia

We investigated inter-annual variation of canopy CO₂ exchange (NEE) and evapotranspiration during a 7-year period over a lowland Dipterocarp forest in Pasoh, Peninsular Malaysia, using the eddy covariance method. Annual rainfall fluctuated between 1,451 and 2,235?mm during this period. Annual evapotranspiration estimated by energy budget correction and gap filling using the relationship between latent heat and available energy was 1,287?±?52?mm. Despite inter-annual variation in rainfall, annual evapotranspiration was stable, except for a slight decrease in the driest year (2009). Evapotranspiration was roughly related to the amount of available energy, but was regulated by stomatal closure to prevent excessive water loss at high vapour pressure deficit. Even during dry periods, no significant decrease in evapotranspiration occurred, as water was supplied from soil layers deeper than 0.5?m. Ecosystem respiration (RE) increased with soil water content. Daytime NEE was also stable during the 7?years, despite climate variability. Afternoon inhibition of canopy photosynthesis was seen every month. Daytime NEE did not become more negative with increasing solar radiation, or with increasing soil water content. During dry periods, gross primary production (GPP) and thus canopy gross photosynthesis decreased slightly, coupled with decreased daytime RE. In this forest, variability in rainfall pattern resulted in seasonal and inter-annual variability in micrometeorology; evapotranspiration, photosynthesis, and RE responded to these changes, and compensated for each other and/or other components of micrometeorology, resulting in rather stable annual evapotranspiration and NEE, even during a very dry year associated with an El Nino Southern Oscillation (ENSO) event.     

Canine histiocytic sarcoma cell lines with SHP2 p.Glu76Gln or p.Glu76Ala mutations are sensitive to allosteric SHP2 inhibitor SHP099

Some canine cases of histiocytic sarcoma (HS) carry an activating mutation in the src homology two domain‐containing phosphatase 2 (SHP2) encoded by PTPN11. SHP099 is an allosteric inhibitor of SHP2 that stabilizes SHP2 in a folded, auto‐inhibited conformation. Here, we examined the expression and mutation status of SHP2 in five canine HS cell lines and evaluated the growth inhibitory properties of SHP099 against these cell lines. All five of the canine HS cell lines expressed SHP2, with three of the lines each harbouring a distinct mutation in PTPN11/SHP2 (p.Glu76Gln, p.Glu76Ala and p.Gly503Val). In silico analysis suggested that p.Glu76Gln and p.Glu76Ala, but not p.Gly503Val, promote shifting of the SHP2 conformation from folded to open‐active state. SHP099 potently suppressed the growth of two of the mutant cell lines (harbouring SHP2 p.Glu76Gln or p.Glu76Ala) but not that of the other three cell lines. In addition, SHP099 suppressed ERK activation in the cell line harbouring the SHP2 p.Glu76Ala mutation. The SHP2 p.Glu76Gln and p.Glu76Ala mutations are considered to be activating mutations, and the signal from SHP2 p.Glu76Ala is inferred to be transduced primarily via the ERK pathway. Moreover, SHP099‐sensitive HS cells, including those with SHP2 p.Glu76Gln or p.Glu76Ala mutations, may depend on these mutations for growth. Therefore, targeting cells harbouring SHP2 p.Glu76Gln and p.Glu76Ala with SHP099 may be an approach for the treatment of canine HS.     

Identification and Biochemical Characterization of Halisulfate 3 and Suvanine as Novel Inhibitors of Hepatitis C Virus NS3 Helicase from a Marine Sponge

Hepatitis C virus (HCV) is an important etiological agent that is responsible for the development of chronic hepatitis, liver cirrhosis, and hepatocellular carcinoma. HCV nonstructural protein 3 (NS3) helicase is a possible target for novel drug development due to its essential role in viral replication. In this study, we identified halisulfate 3 (hal3) and suvanine as novel NS3 helicase inhibitors, with IC₅₀ values of 4 and 3 µM, respectively, from a marine sponge by screening extracts of marine organisms. Both hal3 and suvanine inhibited the ATPase, RNA binding, and serine protease activities of NS3 helicase with IC₅₀ values of 8, 8, and 14 µM, and 7, 3, and 34 µM, respectively. However, the dengue virus (DENV) NS3 helicase, which shares a catalytic core (consisting mainly of ATPase and RNA binding sites) with HCV NS3 helicase, was not inhibited by hal3 and suvanine, even at concentrations of 100 µM. Therefore, we conclude that hal3 and suvanine specifically inhibit HCV NS3 helicase via an interaction with an allosteric site in NS3 rather than binding to the catalytic core. This led to the inhibition of all NS3 activities, presumably by inducing conformational changes.     

Soil physical properties and preferential flow pathways in tropical rain forest, Bukit Tarek, Peninsular Malaysia

Soil physical properties and water movement within soil were investigated using dyes in a tropical rain forest, the Bukit Tarek Experimental Watershed of Peninsular Malaysia. The saturated hydraulic conductivity (K _s) decreased with increasing soil depth. TheK _s values were higher than those reported for other tropical soils. The geometric means of theK _s values ranged from 4.69×10⁻³ (80 cm) to 4.07×10⁻² cm s⁻¹ (10cm). This suggests saturation overland flow may not be dominant but that subsurface flow must play an important role in stormflow generation. The shapes of the soil moisture characteristic curves resembled those of forest soils which have large changes in volumetric water content at pressure heads <30 cmH₂O. The relatively high conductivities were due to the presence of a porous zone of decomposed root channels which existed continuously in vertical direction. Besides decayed roots, living roots also encourage preferential flow in vertical and lateral (downslope) directions. Termite activities may also form water flow pathways in tropical regions. These detailed results help us analyze water flow within the soil in tropical rain forests.     

Attachment of Malassezia pachydermatis to the ear dermal cells in canine otitis externa.

To investigate the predominance of Malassezia pachydermatis (M. pachydermatis) as a causative agent of canine otitis externa, ear cerumen samples were observed for adhesion of M. pachydermatis to the cornified epithelial cells by light and electron microscopes. The yeasts appeared not to adhere to the cornified epithelial cells directly, but they seemed to exist in the proximity of the epithelial cells with an electron opaque halo-like space around them. Polysaccharide and lipid staining techniques were conducted to identify the substances existing in that space. Lipid substances, not saccharides, were observed around the yeasts and the cornified epithelial cells. These results suggested that in the canine ear canal malassezia yeast attachment to the cornified epithelial cells is mediated by lipids.     

Quantification of genetically modified soybean by quenching probe polymerase chain reaction

Quenching probe (QProbe) polymerase chain reaction (PCR) is a simple and cost-effective real-time PCR assay in comparison with other real-time PCR assays such as the TaqMan assay. We used QProbe-PCR to quantify genetically modified (GM) soybean (Roundup Ready soybean). We designed event-specific QProbes for Le1 (soy endogenous gene) and RRS (recombinant gene), and we quantified certified reference materials containing 0.1, 0.5, 1, 2, and 5% GM soybean. The TaqMan assay was also applied to the same samples, and the results were compared. The accuracy of QProbe-PCR was similar to that of TaqMan assay. When GM soybean content was 0.5% or more, the relative standard deviations of QProbe-PCR were less than 20%. QProbe-PCR is sensitive enough to monitor labeling systems and has acceptable levels of accuracy and precision.     

First record of Cryptosporidium infection in a raccoon dog (Nyctereutes procyonoides viverrinus)

Cryptosporidium species have been found in more than 150 species of mammals, but there has been no report in raccoon dogs. Here we found the Cryptosporidium organism in a raccoon dog, Nyctereutes procyonoides viverrinus, and identified this isolate using PCR-based diagnostic methods. Cryptosporidium diagnostic fragments of the 18S ribosomal RNA, Cryptosporidium oocyst wall protein and 70-kDa heat shock protein genes were amplified from the isolate and sequenced to reveal the phylogenetic relationships between it and other Cryptosporidium species or genotypes reported previously. The results showed that the raccoon dog isolate represented the C. parvum cattle genotype which could be a causative agent in human cryptosporidiosis.