A microaliquoting technique for precise histological annotation and optimization of cell content in frozen tissue specimens

Knowledge of the exact cell content of frozen tissue samples is of growing importance in genomic research. We developed a microaliquoting technique to measure and optimize the cell composition of frozen tumor specimens for molecular studies. Frozen samples of 31 mesothelioma cases were cut in alternating thin and thick sections. Thin sections were stained and evaluated visually. Thick sections, i.e., microaliquots, were annotated using bordering stained sections. A range of cellular heterogeneity was observed among and within samples. Precise annotation of samples was obtained by integration and compared to conventional single face and “front and back” section estimates of cell content. Front and back estimates were more highly correlated with block annotation by microaliquoting than were single face estimates. Both methods yielded discrepant estimates, however, and for some studies may not adequately account for the heterogeneity of mesothelioma or other malignancies with variable cellular composition. High yield and quality RNA was extracted from precision annotated, tumor-enriched subsamples prepared by combining individual microaliquots with the highest tumor cellularity estimates. Microaliquoting provides accurate cell content annotation and permits genomic analysis of enriched subpopulations of cells without fixation or amplification.     

Differential cell surface properties of vegetative Bacillus

Aims:  The genus Bacillus encompasses a wide range of species which display varying pathogenic abilities. The hydrophobicity of a range of Bacillus species was determined to evaluate the correlation between bacterial hydrophobicity and pathogenicity.
Methods and Results:  Bacterial adhesion to hydrocarbon assays were used to determine the hydrophobicity of various Bacillus species. Significant differences in the hydrophobicity of vegetative Bacilli were found. Specifically, vegetative Bacillus anthracis or Bacillus thuringiensis cells were highly hydrophobic whereas Bacillus cereus or Bacillus subtilis were only slightly hydrophobic using this test. Cell adhesion assays using A549 or J774 cells were used to demonstrate a correlation between the bacterial hydrophobicity profiles with the ability to adhere to the mammalian cell lines.
Conclusions:  The ability of Bacillus species to adhere to mammalian cell lines correlates with the hydrophobicity of the bacteria and also correlates with the relative pathogenicity of some of the Bacillus species tested.
Significance and Impact of the Study:  This work suggests that study of the physical-chemical properties of vegetative cells could inform future approaches for the rapid identification and discrimination of potentially pathogenic Bacilli .     

A microaliquoting technique for precise histological annotation and optimization of cell content in frozen tissue specimens

Knowledge of the exact cell content of frozen tissue samples is of growing importance in genomic research. We developed a microaliquoting technique to measure and optimize the cell composition of frozen tumor specimens for molecular studies. Frozen samples of 31 mesothelioma cases were cut in alternating thin and thick sections. Thin sections were stained and evaluated visually. Thick sections, i.e., microaliquots, were annotated using bordering stained sections. A range of cellular heterogeneity was observed among and within samples. Precise annotation of samples was obtained by integration and compared to conventional single face and “front and back” section estimates of cell content. Front and back estimates were more highly correlated with block annotation by microaliquoting than were single face estimates. Both methods yielded discrepant estimates, however, and for some studies may not adequately account for the heterogeneity of mesothelioma or other malignancies with variable cellular composition. High yield and quality RNA was extracted from precision annotated, tumor-enriched subsamples prepared by combining individual microaliquots with the highest tumor cellularity estimates. Microaliquoting provides accurate cell content annotation and permits genomic analysis of enriched subpopulations of cells without fixation or amplification.     

Optimization of the cell wall microenvironment allows increased production of recombinant Bacillus anthracis protective antigen from B. subtilis.

The stability of heterologous proteins secreted by gram-positive bacteria is greatly influenced by the microenvironment on the trans side of the cytoplasmic membrane, and secreted heterologous proteins are susceptible to rapid degradation by host cell proteases. In Bacillus subtilis, degradation occurs either as the proteins emerge from the presecretory translocase and prior to folding into their native conformation or after the native conformation has been reached. The former process generally involves membrane- and/or cell wall-bound proteases, while the latter involves proteases that are released into the culture medium. The identification and manipulation of factors that influence the folding of heterologous proteins has the potential to improve the yield of secreted heterologous proteins. Recombinant anthrax protective antigen (rPA) has been used as a model secreted heterologous protein because it is sensitive to proteolytic degradation both before and after folding into its native conformation. This paper describes the influence of the microenvironment on the trans side of the cytoplasmic membrane on the stability of rPA. Specifically, we have determined the influence of net cell wall charge and its modulation by the extent to which the anionic polymer teichoic acid is D-alanylated on the secretion and stability of rPA. The potential role of the dlt operon, responsible for D-alanylation, was investigated using a Bacillus subtilis strain encoding an inducible dlt operon. We show that, in the absence of D-alanylation, the yield of secreted rPA is increased 2.5-fold. The function of D-alanylation and the use of rPA as a model protein are evaluated with respect to the optimization of B. subtilis for the secretion of heterologous proteins.     

Factors affecting the presence of Typhlodromusspp. (Acarina: Phytoseiidae) in the calyx cavities of apple fruits and implications for integrated pest management

Predatory mites of the family Phytoseiidae include several species of importance as biological control agents of phytophagous mites in various crops including apples. We report on the post-harvest presence of the motiles of two species, Typhlodromus occidentalis and Typhlodromus pyri, in the calyx cavities of apple fruits of three cultivars. The mean numbers of motiles per fruit were as high as 6.10. The phytoseiid densities were lowest in cv. Bonza and tended to be greater in samples taken in late autumn, particularly in cv. Red Delicious. No significant effect (p > 0.05) was observed for different insecticide treatments. Phytoseiids remained present within fruits of cv. Granny Smith left on the orchard floor in winter. Together with an observed reduction in the numbers of T. occidentalis in the early spring, this suggests that fruits constitute an overwintering refuge. The findings are discussed in relation to the implications for biological control, in particular the minimization of the removal of natural enemies from orchards. This revised version was published online in November 2006 with corrections to the Cover Date.     

Design and characterization of novel hybrid antimicrobial peptides based on cecropin A, LL-37 and magainin II

Antimicrobial peptides (AMPs) are a naturally occurring component of the innate immune response of many organisms and can have activity against both Gram-negative and Gram-positive bacterial species. In order to optimize and improve the direct antimicrobial effect of AMPs against a broad spectrum of bacterial species, novel synthetic hybrids were rationally designed from cecropin A, LL-37 and magainin II. AMPs were selected based on their α-helical secondary structure and fragments of these were analyzed and combined in silico to determine which hybrid peptides would form the best amphipathic cationic α-helices. Four hybrid peptides were synthesized (CaLL, CaMA, LLaMA and MALL) and evaluated for direct antimicrobial activity against a range of bacterial species (Bacillus anthracis, Burkholderia cepacia, Francisella tularensis LVS and Yersinia pseudotuberculosis) alongside the original 'parent' AMPs. The hybrid peptides showed greater antimicrobial effects than the parent AMPs (in one case a parent is completely ineffective while a hybrid based on it removes all traces of bacteria by 3h), although they also demonstrated higher hemolytic properties. Modifications were then carried out to the most toxic hybrid AMP (CaLL) to further improve the therapeutic index. Modifications made to the hybrid lowered hemolytic activity and also lowered antimicrobial activity by various degrees. Overall, this work highlights the potential for rational design and synthesis of improved AMPs that have the capability to be used therapeutically for treatment of bacterial infections.     

Production of Bacillus anthracis protective antigen is dependent on the extracellular chaperone,PrsA

Protective antigen (PA) is a component of the Bacillus anthracis lethal and edema toxins and the basis of the current anthrax vaccine. In its heptameric form, PA targets host cells and internalizes the enzymatically active components of the toxins, namely lethal and edema factors. PA and other toxin components are secreted from B. anthracis using the Sec-dependent secretion pathway. This requires them to be translocated across the cytoplasmic membrane in an unfolded state and then to be folded into their native configurations on the trans side of the membrane, prior to their release from the environment of the cell wall. In this study we show that recombinant PA (rPA) requires the extracellular chaperone PrsA for efficient folding when produced in the heterologous host, B. subtilis; increasing the concentration of PrsA leads to an increase in rPA production. To determine the likelihood of PrsA being required for PA production in its native host, we have analyzed the B. anthracis genome sequence for the presence of genes encoding homologues of B. subtilis PrsA. We identified three putative B. anthracis PrsA proteins (PrsAA, PrsAB, and PrsAC) that are able to complement the activity of B. subtilis PrsA with respect to cell viability and rPA secretion, as well as that of AmyQ, a protein previously shown to be PrsA-dependent.     

Field evaluation of the effects of the insect growth regulator tebufenozide on entomophagous arthropods and pests of apples

Organophosphate insecticides are very widely used in commercial apple production to control fruit-attacking pests but their broad-spectrum activity constrains biological control of other pests. Compounds with narrow-spectrum activity are therefore desirable. The insect growth regulator (IGR) tebufenozide was compared with another IGR, fenoxycarb, and the organophosphate, azinphos-methyl, in a replicated field trial in the 1994/1995 apple-production season. Vacuum sampling of the tree foliage on five occasions during the growing season showed significantly lower populations of various natural enemies (spiders, lacewings and the specialist mite predator Stethorus spp. adults and larvae) in the azinphos-methyl treatment than in either of the two IGR treatments. The two-spotted mite ( Tetranychus urticae Koch) was most numerous in the azinphos-methyl treatment. In 1995/1996, the entire trial area was placed under a tebufenozide treatment program to determine the extent to which natural enemy populations would recover when broad-spectrum insecticide (azinphos-methyl) use was halted. Populations of polyphagous natural enemies assumed levels broadly equivalent to those observed under IGR treatments the previous year. Numbers of Stethorus spp. were lower than in the 1994/1995 season, possibly because T. urticae (prey) populations were much reduced from the previous season's densities. All three insecticide treatments were equally effective in controlling the lepidopteran pests, codling moth ( Cydia pomonella (L.)), lightbrown apple moth ( Epiphyas postvittana (Walker)) and early season caterpillars (predominantly Helicoverpa punctigera (Wallengren)). Results indicate that tebufenozide provides good control of lepidopteran pests, while allowing the rapid build-up of natural enemies which contribute to control of other pests.     

Control of codling moth in small plots by mating disruption: alone and with limited insecticide

Mating disruption alone and mating disruption supplemented with limited applications of either azinphos-methyl or fenoxycarb was evaluated in 0.11–0.30 ha plots for the control of codling moth Cydia pomonella (L.) (Tortricidae: Olethreutinae) populations.Where populations were low, mating disruption alone was sufficient to keep codling moth damage levels below 1% at harvest. Low was defined as no more than 0.2% damage at harvest and fewer than 1.3 larvae/metre of trap band from January–March inclusive in the previous season. Neither mating disruption alone nor when supplemented with azinphos-methyl or fenoxycarb during the first spring generation gave commercially acceptable levels of control in other than low density populations. Small plot size may have contributed to the failures.At the high density site unintentional supplementary control provided by drift of azinphos-methyl from border areas to which it was applied throughout the season gave excellent control in the first year and suggested an interaction between low dosages of insecticide and pheromone treatments that enhanced the disruptive effect of the latter.     

Tebufenpyrad: compatibility with integrated mite control on apples

Successful integrated mite control (IMC) depends on the availability of acaricides which act selectively on pest mites with minimal effects on predators. Laboratory bioassays of tebufenpyrad against adult female Panonychus ulmi (Koch) provided baseline toxicity data and showed it to be highly active. Bioassays also showed tebufenpyrad was toxic to the predatory mite Typhlodromus pyri Scheuten but it was approximately 70 times less toxic to the predators than to P. ulmi at the lc ₅₀. Field trials using four concentrations of tebufenpyrad (10, 7.5, 5 and 2.5 g a.i./100 l) confirmed it was selectively more toxic to the pest mites than to predators and showed that it is compatible with IMC. It is suggested that a concentration of 5 g a.i./100 l would be suitable for IMC. A resistance management strategy for tebufenpyrad is proposed.