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.     

Multiple myeloma in 16 cats: a retrospective study

BACKGROUND: There is limited published information regarding feline multiple myeloma. Diagnostic criteria are derived from canine studies and to our knowledge, have not been critically reviewed for cats. OBJECTIVE: To evaluate the clinical and laboratory findings in cats with multiple myeloma and appraise diagnostic criteria. METHODS: Retrospective evaluation of medical records was performed. Inclusion required an antemortem diagnosis of multiple myeloma using 2 of 4 criteria: 1) >or=20% plasma cells in the bone marrow, or >or=10% if atypical plasma cells; 2) paraproteinemia; 3) radiographically-evident osteolysis; 4) light chain proteinuria. Alternatively, a postmortem diagnosis was based on the findings of multiple plasma cell neoplasms, with marrow involvement. RESULTS: Sixteen cats were diagnosed with multiple myeloma between 1996 and 2004, with a median age of 14.0 years; 9 of 16 (56%) were castrated males, and 7 of 16 (44%) were spayed females. Laboratory abnormalities included hyperglobulinemia (14/16, 87.5%), with 11/14 (78.5%) monoclonal and 3/14 (21.4%) biclonal gammopathies; hypoalbuminemia (4/16, 25%); light chain proteinuria, (4/9, 44.4%); hypocholesterolemia (11/16, 68.7%); hypercalcemia, (3/15, 20%); nonregenerative anemia, (11/16, 68.7%); regenerative anemia, (1/16, 6.2%); neutropenia (5/15, 33.3%); thrombocytopenia (8/16, 50%); and marrow plasmacytosis (14/15, 93.3%). Plasma cells were markedly immature, atypical, or both in 10 of 12 (83.3%) cats. Focal or multifocal osteolysis was noted in 6 of 12 (50%) cats for which radiographs were available for review; generalized osteopenia was found in 1 (8.3%) cat. Noncutaneous, extramedullary tumors were found in all cats assessed, 7/7 (100%), including spleen (6), liver (3), and lymph nodes (4). The disease in 1 of 2 cats with cutaneous tumors progressed to plasmacytic leukemia. CONCLUSIONS: Common findings in feline multiple myeloma include atypical plasma cell morphology, hypocholesterolemia, anemia, bone lesions, and multi-organ involvement. Based on the results of this study, we advocate modifying diagnostic criteria in cats to include consideration of plasma cell morphology and visceral organ infiltration.     

Derivation and characterisation of a live equid herpes virus-1 (EHV-1) vaccine to protect against abortion and respiratory disease due to EHV-1

A German abortion isolate of EHV-1 (strain M8) was grown in equine dermal (ED) cells at a low multiplicity of infection in presence of 5-bromo-2-deoxy uridine. The resulting stock was dialysed, titrated and cloned by terminal dilution in ED cells grown in 96-well microtitration plates. Of 192 clones each originating from a single focus, clone 147 (C147) was found to be restricted for growth at and above temperatures of 38.5 degrees C. It was also restricted for growth at 37 degrees C in rabbit kidney (RK-13) cells which are widely used for the isolation and titration of EHV-1; hence clone 147 was EHV-4-like.Clone 147 showed a remarkable efficacy as a vaccine in protecting conventional pregnant Welsh Mountain pony mares against abortions due to EHV-1. A single intranasal (IN) vaccination protected five out of six (83.3%), and four out of five (80%) of mares upon challenge 4 and 5-6 months, respectively, after the immunisation, whereas all six unvaccinated mares aborted between 9 and 19 days after IN EHV-1 challenge. With the exception of the day 9 abortion, foetuses of the remaining five mares were EHV-1 infected. Placenta from the early aborting mare was, however, EHV-1 positive. Both groups of vaccinated mares were also significantly protected against clinical reaction (notably pyrexia), nasal shedding and viraemia following challenge infection.     

Secondary plant metabolites as targets for genetic modification of crop plants for pest resistance

Three genetic manipulation strategies are described for enhancing the effectiveness of secondary metabolites of crop plants in reducing pest and disease damage. Targets for gene transfer studies are secondary metabolites of oilseed rape, lepidopteran sex pheromones and drimane anti-feedants. Initial work towards isolation of genes for the biosynthesis of drimane anti-feedants is described.     

Cornification defect in the golden retriever: clinical, histopathological, ultrastructural and genetic characterisation

Veterinarians have recognised a nonpruritic skin disease in the Golden retriever breed characterised by excessive scaling of large, variably pigmented flakes of skin in otherwise healthy dogs. This prospective case series describes clinical, histopathological, ultrastructural and genetic features of this cornification defect in 17 affected dogs. The condition affects young dogs of either sex and is characterised by symmetrical, predominantly ventro-lateral scaling and hyperpigmentation of the trunk. Histopathological and ultrastructural changes of the stratum corneum are suggestive of delayed degradation of corneodesmosomes. A genetic aetiology is proposed and a single-trait autosomal recessive mode of inheritance discussed.     

A novel medium devoid of ruminant peptone for high yield growth of Mycoplasma ovipneumoniae

Mycoplasma ovipneumoniae is considered an emerging veterinary pathogen causing pneumonia in sheep and goats worldwide. Currently it has not been possible to define a growth medium that yields the maximum growth of M. ovipneumoniae within a short incubation period. Growth yields of M. ovipneumoniae in Eaton's medium are variable and not as consistently high as those seen with other Mycoplasma spp. This study investigated the ability of different M. ovipneumoniae field strains to grow in various media formulations, where PPLO broth was replaced by a vegetable protein source, and comparisons were made in terms of strain viability in Eaton's medium. Studies were also conducted to determine the optimal carbohydrate source for use in the M. ovipneumoniae medium. Generally, it was found that different strains showed good growth in all media tested, with growth yields at 24h in TSB-1 medium higher than those observed with Eaton's medium. Growth yields reached 10(8) to 10(9)cfu ml(-1) within 24h for particular field strains, with all strains achieving this growth level within 48-72h.     

Evaluation of dietary protein requirement for the growth performance of minor carp,Cirrhinus reba (Hamilton, 1822) fingerlings

An experiment was designed to evaluate the requirement of dietary protein for Reba carp, Cirrhinus reba fingerlings using semi‐purified diet. Five isocaloric diets were prepared containing 20%, 25%, 30%, 35% and 40% level of protein using casein, gelatin as a major protein source. The experiment was conducted for 105 days in triplicates to evaluate the growth, survival, feed utilization, carcass composition of C. reba. Mean weight gain was highest (p < .05) in T3. Specific growth rate was highest in T3 and lowest in T1. Similarly, significantly (p < .05) highest net fish yield were recorded in T3. No significant difference was recorded in survivability. Feed conversion ratio, protein efficiency ratio, feed conversion efficiency and protein conversion efficiency were significantly (p < .05) higher for T3 compared with other treatments. Highest carcass protein and lipid values were recorded in T3. So, conclusion can be drawn that 30% crude protein is optimum for overall performance. Furthermore, broken‐line analysis for specific growth rate, final biomass and net fish yield indicated that dietary protein level of 28.148 to 28.253% is optimum for the best growth performance of fingerling C. reba.     

Proanthocyanidin-rich extracts from cranberry fruit (Vaccinium macrocarpon Ait.) selectively inhibit the growth of human pathogenic fungi Candida spp. and Cryptococcus neoformans

Cranberry ( Vaccinium macrocarpon ) has been shown in clinical studies to reduce infections caused by Escherichia coli and other bacteria, and proanthocyanidins are believed to play a role. The ability of cranberry to inhibit the growth of opportunistic human fungal pathogens that cause oral, skin, respiratory, and systemic infections has not been well-studied. Fractions from whole cranberry fruit were screened for inhibition of five Candida species and Cryptococcus neoformans , a causative agent of fungal meningitis. Candida glabrata , Candida lusitaniae , Candida krusei , and Cryptococcus neoformans showed significant susceptibility to treatment with cranberry proanthocyanidin fractions in a broth microdilution assay, with minimum inhibitory concentrations as low as 1 μg/mL. MALDI-TOF MS analysis of subfractions detected epicatechin oligomers of up to 12 degrees of polymerization. Those containing larger oligomers caused the strongest inhibition. This study suggests that cranberry has potential as an antifungal agent.