Validation and application of a high fidelity mRNA linear amplification procedure for profiling gene expression

The need for microgram quantities of RNA for microarray experiments has hindered application of this novel technology in cell types/tissue samples with limited abundance of RNA. In this study, potential application of T7-based linear RNA amplification was investigated for use in gene expression profiling experiments where starting material is limited. Yield and integrity of amplified antisense RNA (aaRNA), microarray hybridization intensities, and fidelity of differential gene expression detected were determined for arrays generated for unamplified versus amplified RNA from the same homogenous starting pools. Total RNA was extracted from bovine spleen and fetal ovary, serially diluted to concentrations ranging from 2 microg to 500 pg and amplified. Quality and quantity of total input RNA and aaRNA were assessed by spectrophotometry, gel electrophoresis and bioanalyzer. In experiment 1, we determined the optimal amounts of aaRNA generated from 20, 40, 200 ng and 2 microg input total RNA for use in cDNA synthesis, labeling and array hybridization that would yield robust and consistent hybridization signals on a bovine oocyte cDNA microarray. In experiment 2, comparison of microarray hybridization intensities and fidelity of differential gene expression between aaRNA generated from 2, 20 and 40 ng input total RNA versus unamplified RNA (uRNA) were conducted. The hybridization intensities for each of the 7000 spots per slide for microarrays conducted using aaRNA versus uRNA were highly correlated (2 ng = 0.84, 20 ng = 0.88, 40 ng = 0.90; P < 0.01). The false positive rate was low and similar (4.0% versus 4.4%) for arrays done with uRNA and aaRNA. Ninety-seven ESTs were detected as differentially expressed in the fetal ovary versus spleen at > 1.5- or < 0.5-fold using uRNA (P < 0.05). However, the number of genes detected in arrays using aaRNA was approximately 1.5-2.5 times greater than with uRNA. Approximately, 65-70% of differentially expressed genes were common between uRNA and aaRNA arrays. Relative fold-expression (Cy3/Cy5 ratios) for 25 overlapping abundant genes was comparable for uRNA versus aaRNA arrays with 2 and 20 ng total RNA as input. Results demonstrate that T7-based linear amplification of small amounts of input RNA and use of aaRNA in microarray experiments retains fidelity of detection of differential gene expression that is relatively comparable to experiments done with uRNA and provides a potentially viable approach to facilitate gene expression profiling using limited amounts of starting material.     

A multi-state Salmonella Typhimurium outbreak associated with frozen vacuum-packed rodents used to feed snakes

From December 2005 through January 2006, the Minnesota Department of Health (MDH) identified four human clinical isolates of Salmonella Typhimurium that were indistinguishable by pulsed-field gel electrophoresis (PFGE). During routine interviews, three of the cases reported attending the same junior high school and two handled snakes in the science classroom. MDH collected environmental samples from the school's science classroom for Salmonella culturing; these included environmental samples and frozen vacuum-packed mice purchased over the internet to feed the classroom snakes. Through PulseNet, a national molecular subtyping surveillance network for enteric bacteria, 21 human S. Typhimurium isolates with indistinguishable PFGE patterns were identified in the United States since December 2005. Each state determined whether these human cases had recent exposure to snakes fed vacuum-packed rodents. Texas state officials conducted tracebacks of the vacuum-packed mice and collected samples at the breeding facility. Nineteen of 21 cases were interviewed, and seven reported contact with frozen vacuum-packed rodents from the same internet-based supplier in Texas. In Minnesota, the outbreak PFGE subtype of S. Typhimurium was isolated from the snakes, frozen feed rodents, and the classroom environment. Three human cases were identified in Michigan, Pennsylvania, and Wyoming. The outbreak PFGE subtype of S. Typhimurium was isolated from the Pennsylvania case's frozen rodents and the Michigan case's pet snake. The outbreak PFGE subtype of S. Typhimurium was also isolated from the supplier's rodent facility. This was a S. Typhimurium outbreak associated with frozen rodents. Human transmission likely occurred through direct contact with snakes and contaminated environmental surfaces. This report represents the second recent multi-state salmonellosis outbreak associated with commercially distributed rodents. Stronger oversight of the commercial rodent industry is warranted.     

Wireworm damage reduction in potatoes with an attract-and-kill strategy using <Emphasis Type="Italic">Metarhizium brunneum</Emphasis>

Innovative wireworm control strategies are required to implement integrated pest management on the basis of the (EC) No regulation 1107/2009 and Directive 2009/128/EC. Entomopathogenic fungi, such as Metarhizium brunneum (Metschnikoff) Sorokin, are potential biological control agents for wireworm control but do not achieve high control efficacies in the field when applied as a conidia suspension. In a 2-year study, wireworm control with a novel attract-and-kill strategy aimed at enhancing M. brunneum efficacies in organic potato production systems in Lower Saxony, Germany. The approach is based on the attraction of wireworms (Agriotes spp. Eschscholtz) towards an artificial carbon dioxide-emitting source, using baker’s yeast (Saccharomyces cerevisiae Meyen ex Hansen) in combination with M. brunneum conidia for wireworm infection. Both components were encapsulated in alginate as a carrier material and applied in a mixture with two types of beads (one for encapsulated yeast and one for M.brunneum conidia). An application of these beads within the potato rows during potato planting reduced wireworm tuber damage by 37–75% relative to the untreated control and was able to enhance the efficacy of M. brunneum by up to 35% through an attract-and-kill approach compared to beads without a carbon dioxide source only. This strategy offers a high potential to promote biological wireworm control as an alternative to insecticide use by potentially reducing the inoculum compared to an inundate M. brunneum conidia release strategy.     

Long-term realised and projected growth impacts caused by autumn gum moth defoliation of 2-year-old Eucalyptus nitens plantation trees in Tasmania,Australia

Insect damage to production forests has the potential to reduce financial returns by retarding tree growth and causing mortality, however, long-term realised quantification of these losses is rare. In order to help elucidate economic damage thresholds for making spray decisions we capitalised on a natural outbreak of autumn gum moth, Mnesampela privata, in a 2-year-old Eucalyptus nitens plantation. Following the partial chemical control of this insect outbreak we measured the tree growth variables diameter at breast height over bark and height of five differing tree defoliation classes for 75 months following tree damage. At the end of this period a threshold model was fitted to describe the relationship between tree defoliation and realised tree wood volumes. The model revealed that realised stand wood volume was not significantly affected up until defoliation exceeded 60% and then declined sharply after this defoliation level was reached. Further support for this defoliation threshold was evident from multiple comparisons among defoliation classes that showed 50% defoliated trees did not have significantly different wood volume compared to more lightly defoliated trees, but did have significantly greater wood volume compared to trees that were 72% or more defoliated. To determine if the realised differences in wood volume resulted in differences in yield over a plantation rotation the E. nitens growth model NITGRO was used to on-grow trees to age 15 years for a ‘best case’ (type 1 growth response, constant growth rates from last inventory until harvest) and ‘worst case’ (type 2 growth response, divergent growth rates from last inventory until harvest) scenario. The threshold model was then fitted to the outcomes of both scenarios and the economic consequences of defoliation were clearly dependent on the growth function assumed.     

印度西部5种热带干落叶林中凋落物中生物元素和返回到绿叶中的季节性变化(英文)

本文对印度西部5种热带干落叶林中凋落物里生物元素的季节性变化和它们返回到新鲜叶中季节性变化进行研究。对5个树种:阿拉伯金合欢(Acacia nilotica L.)、金合欢(Acacia leucophloea Roxb.)、柚木(Tectonagrandis L.f.)、绒毛野独活(Miliusa tomentosa Roxb.)和紫矿(Butea mono-sperma Lain.)中凋落物的成熟叶、枝和再生部分的N、P、Ca、Mg、K和Na的浓度,以及老叶和幼叶中浓度间的关系进行调查。对这些树种的总干物质进行记录,并分析它们所含的N、P、Ca、Mg、K和Na的含量。其中阿拉伯金合欢叶子中N的含量最高,而金合欢的叶子里Ca和Mg含量最高。叶中含P最多的是阿托伯金合欢、金合欢和紫矿,而含P最少的是柚木和绒毛野独活。K和Na含量在这些树种中没有明显的区别。观察发现,N、P和K的浓度随季节变化大,但Ca和Mg则不然。唯有钾在所有树种中都是最稳定的,吸收N和P可以用于来年新叶的生长。从老叶和凋落物中吸收的营养会支持新叶的生长,并增加土壤的肥力。图1表4参30。     

Restriction of Starch Granule Swelling by Iodine During Heating

Differences in the degree of starch granule swelling and granule morphology are shown as a function of iodine concentration during heating. The observations reveal a restriction and delay in wheat and corn starch swelling in presence of iodine (0.02%) and a lack of swelling at higher iodine concentration (0.2%). The presence of iodine during heating did not influence waxy corn starch granule swelling, even at the higher concentration. A delay in the increase in paste viscosity during heating was observed, and gel formation was precluded after cooling at the higher iodine concentration. Waxy corn starch pastes formed a weak gel even at the higher iodine concentration. Spectrophotometric analyses showed that polymers leach into the solution when heated in the presence of 0.02% iodine, while no leaching was observed at 0.2% iodine concentration. Furthermore, the length and the amount of polymers leaching from normal corn were different from that observed for wheat starch. Thermal analyses reveal a shift in the onset of gelatinization temperature and an increase in the enthalpy in the amylose‐lipid region of the endotherm. While the iodine‐polymer complex did not appear to exhibit an endotherm during heating, the granules exhibited a diffused polarized cross, suggesting the presence of an ordered complex.     

Acidification of Surface Water in Central India

The results of the seasonal analysis of the chemistry of rain, fog and surface water of central India for the two years: June 1996 to May 1998 are presented. A total of 636 rain water samples, 146 fog water samples and 226 surface water samples of 8 locations: namely, Ambikapur, Baikunthpur, Korba, Bilaspur, Raipur, Bhilai, Dallirajhara and Kanker, from different sites were collected for the present study. This paper documents the occurrence of acidic rain and fog events and their effect on surface water pH and aquatic life in central India. Most of the rain and fog water samples collected from Baikunthpur and Korba sites were found to be acidic in nature (i.e. pH < 5). The volume weighted mean pH of rain and fog water samples of these sites was 4.8 and 4.4 with lowest pH 4.4 and 4.0, respectively. However, samples from Ambikapur, Bilaspur, Raipur and Bhilai were slightly acidic and their pH values were always around 5.8. One reservoir (mean pH 5.8) and two stop dams (mean pH 4.4 and 4.2) in Baikunthpur area were found to be acidic. The effect of acidification of water on metal-accumulation in muscles of fishes was studied. Concentrations of Al, Hg, Mn, Cd and Pb were higher in fish from acid, than from less acid waters. Causes of acidification of the atmospheric water and surface water are critically discussed.     

Effect of Sources and Levels of Zinc on the Yield and Composition of Rice (Oryza sativa) on Alfisol under Field Condition

Significant increase in yield of rice and straw was obtained with zinc application during two years of experimentation. Among different sources tried, zinc sulphate proved better. An increase in the concentration and uptake of zinc and nitrogen was found with the application of zinc, whereas there was a reverse trend in the concentration and uptake of phosphorus and potassium.     

Explaining photodermatosis: cyclopentenone vs. alpha-methylene-gamma-lactone natural products.

The possible role of cyclopentenone-containing sesquiterpene lactones in the cause of photochemical chronic actinic dermatitis (CAD) is examined in light of recent reports that the alpha-methylene-gamma-lactone group of these natural products forms 2+2 photoadducts with the DNA base thymine. Neither cyclopentenone nor tenulin (a cyclopentenone-containing sesquiterpene lactone) form such photoadducts with thymine either with sunlight or a UV lamp (300 nm). In contrast, alpha-methylenebutyrolactone readily forms the 2+2 photoadduct with thymine in sunlight. Thus, the photochemical role of the alpha-methylene-gamma-lactone group (rather than cyclopentenone) is strongly implicated in the CAD disease.     

The location of primary replication of the herpesvirus of bovine malignant catarrhal fever in rabbits

The herpesvirus of malignant catarrhal fever (MCFV) was isolated from the spleen of $\text{2}\text{3}$ rabbits 4 days after the intravenous inoculation of infectious lymph node suspension, while no virus could be isolated at 2 and 6 days post inoculation (p.i.). Indirect immunofluorescence identified the antigen-positive cells at 4 days p.i. as medium sized lymphocytes lying in the venous sinuses of the spleen, lower numbers of fluorescing cells being seen in the paracortical areas of lymph nodes and in the thymus. Scanty fluorescence, without isolation of virus, was evident in the spleen at 6 days p.i. but by day 8 p.i. virus could be isolated irregularly from spleen and lymph nodes and at 12 days p.i. was found in all lymphoid tissues in those rabbits with lymphadenitis and pyrexia. The primary replication in the spleen at 4 days is compared with other herpes induced lymphoproliferative disorders.