Improve protective efficacy of a TB DNA-HSP65 vaccine by BCG priming

Vaccines are considered by many to be one of the most successful medical interventions against infectious diseases. But many significant obstacles remain, such as optimizing DNA vaccines for use in humans or large animals. The amount of doses, route and easiness of administration are also important points to consider in the design of new DNA vaccines. Heterologous prime-boost regimens probably represent the best hope for an improved DNA vaccine strategy. In this study, we have shown that heterologous prime-boost vaccination against tuberculosis (TB) using intranasal BCG priming/DNA-HSP65 boosting (BCGin/DNA) provided significantly greater protection than that afforded by a single subcutaneous or intranasal dose of BCG. In addition, BCGin/DNA immunization was also more efficient in controlling bacterial loads than were the other prime-boost schedules evaluated or three doses of DNA-HSP65 as a naked DNA. The single dose of DNA-HSP65 booster enhanced the immunogenicity of a single subcutaneous BCG vaccination, as evidenced by the significantly higher serum levels of anti-Hsp65 IgG2a Th1-induced antibodies, as well as by the significantly greater production of IFN-γ by antigen-specific spleen cells. The BCG prime/DNA-HSP65 booster was also associated with better preservation of lung parenchyma. The improvement of the protective effect of BCG vaccine mediated by a DNA-HSP65 booster suggests that our strategy may hold promise as a safe and effective vaccine against TB.     

Hydrosols evaluation in pest control: insecticidal and settling inhibition potential against Myzus persicae (Sulzer)

The hydrosols are by‐products derived during the extraction of essential oils. Although essential oils have been widely evaluated for their insecticidal activities, the possible use of hydrosols in pest control has been almost unknown. The effects of the hydrosols of Origanum majorana (marjoram), Mentha pulegium (pennyroyal), and Melissa officinalis (lemon balm) on the survival and settling behaviour of the aphid pest Myzus persicae were investigated. The hydrosols were isolated using Clevenger hydrodistillation (i) with conventional heating (HD) and (ii) assisted by microwaves (MWHD). GC‐MS analysis showed that the volatiles occurring in the hydrosols were similar between the two techniques. Hydrosols were assayed for possible settling inhibitory effects on M. persicae in Petri dishes (15 cm diameter). In each dish, a sprayed together with an unsprayed eggplant leaf piece was placed. An adult aphid <24 h old was released on the treated leaf and its path length was recorded during the initial 10 min. Then, its position (on the treated or untreated leaf) was recorded 10, 20, 30, 60 min and 24 h after spraying. M. officinalis HD hydrosol resulted in fourfold increase of the path length compared with the control (deionized water). The last observation (24 h) revealed that M. officinalis and M. pulegium HD hydrosols had the strongest inhibitory effect. Additionally, O. majorana hydrosols caused 10–15% aphid mortality after 24 h. In this study, the wider use of the MWHD technique is further supported as in addition to its well‐known advantages (i.e. shorter distillation time, less energy consumption), the effects of the hydrosols produced are comparable to those obtained by HD. Most importantly, the results clearly showed that the potential of hydrosols in pest control ought not to be ignored and should attract the interest of future studies.     

"Silent" sites in Drosophila genes are not neutral: evidence of selection among synonymous codons

The patterns of synonymous codon usage in 91 Drosophila melanogaster genes have been examined. Codon usage varies strikingly among genes. This variation is associated with differences in G+C content at silent sites, but (unlike the situation in mammalian genes) these differences are not correlated with variation in intron base composition and so are not easily explicable in terms of mutational biases. Instead, those genes with high G+C content at silent sites, resulting from a strong "preference" for a particular subset of the codons that are mostly C- ending, appear to be the more highly expressed genes. This suggests that G+C content is reduced in sequences where selective constraints are weaker, as indeed seen in a pseudogene. These and other data discussed are consistent with the effects of translational selection among synonymous codons, as seen in unicellular organisms. The existence of selective constraints on silent substitutions, which may vary in strength among genes, has implications for the use of silent molecular clocks.      

Two levels of resting potential in cardiac purkinje fibers

In an appropriate ionic environment, the resting potential of canine cardiac purkinje fibers may have either of two value. By changing the external K concentration, [K](0), in small steps, it was shown that, in the low (1 mM) Cl, Na-containing solutions used in this study, the two levels of resting potential could be obtained only within a narrow range of [K](0) values; that range was usually found between 1 and 4 mM. Within the critical [K](0) range the resting potential could be shifted from either level to the other by the application of small current pulses. It was shown that under these conditions the steady-state current- voltage relationship was “N-shaped,” and that a region of both negative slope, and negative chord conductance lay between the two stable zero-current potentials. The negative chord conductance was largely due to inward sodium current, only part of which was sensitive to tetrodotoxin (TTX). Under appropriate conditions, the negative chord conductance could be abolished by several experimental interventions and the membrane potential thereby shifted from the lower to the higher resting level: those interventions which were effective by presumably diminishing the steady-state inward current included reducing the external sodium concentration, adding TTX, or adding lidocaine; those which presumably increased the steady-state outward current included small increases in [K](0), brief depolarizations to around -20 mV, or the addition of acetylcholine chloride.     

Nettle extract effects on the aphid Myzus persicae and its natural enemy, the predator Macrolophus pygmaeus (Hem., Miridae)

Abstract:  The effect of water extract of Urtica urens L. on the biological characteristics and population parameters of Myzus persicae (Sulzer) and its natural enemy, the polyphagous predator Macrolophus pygmaeus (Rambur) was investigated on eggplants. Plant material was extracted in deionized water (1 : 5 w/w) for 24 h. Three applications were made at 5-day intervals using the nettle extract, with deionized water as the control. The effect of the extract on survival, duration of nymphal development, pre-reproductive period, fecundity and longevity of both insects was studied at conditions of 25 ± 1°C, 65 ± 5% relative humidity and photoperiod of 16 : 8 h (light : dark). The results showed that the application of nettle extract significantly reduced the fecundity of M. persicae (20% on average) but did not result in a substantial lowering in its intrinsic rate of population increase. However, the predator's biological characteristics and population parameters were not affected by the application of this plant extract. The importance of these results for the use of nettle extract in the management of M. persicae along with its compatibility with the use or conservation of M. pygmaeus is discussed.     

Intratumor heterogeneity of biomarker expression in breast carcinomas

Small biopsy samples are used increasingly to assess the biomarker expression for prognostic information and for monitoring therapeutic responses prior to and during neoadjuvant therapy. The issue of intratumor heterogeneity of expression of biomarkers, however, has raised questions about the validity of the assessment of biomarker expression based on limited tissue samples. We examined immunohistochemically the expression of HER-2neu (p185^erbB-2), epidermal growth factor receptor (EGFR), Bcl-2, p53, and proliferating cell nuclear antigen (PCNA) in 30 breast carcinomas using archived, paraffin embedded tissue and determined the extent of intratumor heterogeneity. Each section was divided into four randomly oriented discrete regions, each containing a portion of the infiltrating carcinoma. For each tumor, the entire lesion and four regions were analyzed for the expression of these markers. Scores of both membrane and cytoplasmic staining of HER-2neu and EGFR, scores of cytoplasmic staining of Bcl-2, and scores of nuclear staining of both p53 and PCNA were recorded. The intensity of staining and the proportion of immunostained cells were determined. A semiquantitative immunoscore was calculated by determining the sum of the products of the intensity and corresponding proportion of stained tumor cells. We analyzed both invasive (IDC) and in situ (DCIS) carcinomas. The Wilcoxon signed-rank test was used for paired comparisons between overall and regional immunoscores and between overall and regional percentages of stained cells. Spearman's correlation coefficients were used to assess the level of agreement of overall biomarker expression with each of the regions. Generalized linear models were used to assess overall and pair-wise differences in the absolute values of percent changes between overall and regional expression of biomarkers. For IDCs, there were no statistically significant differences in the expression of the biomarkers in terms of either the percentage of cells staining or the immunoscores when comparing the entire tumor with each region except for the lower EGFR expression of arbitrarily selected region 1 and lower p53 expression of region 1 compared to that of the entire tumor section. For DCIS, there were no statistically significant differences in the expression of the biomarkers between the entire tumor and each region except in PCNA of region 2 compared to that of entire tumor section. Positive correlation of immunoscores was observed between the entire tumor and each region as well as across all four regions for IDC. Similar observations were noted with DCIS except for HER-2neu and PCNA. No statistically significant differences were observed in the absolute values of percent changes of biomarker expression between overall and the four regions for both DCIS and IDC. Therefore, no significant intratumor heterogeneity in the expression of HER-2neu, Bcl-2, and PCNA was observed in IDC. Minor regional variations were observed for EGFR and p53 in IDC. Similarly, no significant regional variation in the expression of markers was observed in DCIS except for PCNA.     

Discrimination of the closely related biocontrol agents Macrolophus melanotoma (Hemiptera: Miridae) and M. pygmaeus using mitochondrial DNA analysis

The separation of the closely related predatory species Macrolophus melanotoma Costa (= M. caliginosus Wagner) and Macrolophus pygmaeus (Rambur) based exclusively on the different colour pattern of the first antennal segment (white central band in M. melanotoma and entirely black in M. pygmaeus) is rather precarious and their taxonomic status is still in doubt. In the present study their taxonomic status was evaluated by DNA confirmatory analysis and hybridization experiments between M. pygmaeus and a Macrolophus taxon, resembling M. melanotoma, with a first antennal segment entirely black or with a white central band collected from Dittrichia viscosa (L.) W. Greuter (Asteraceae) in southern Greece. Adult females from Dittrichia plants hybridized with males of M. pygmaeus and vice versa did not produce viable eggs. The Macrolophus species from Dittrichia irrespective of the first antennal segment coloration differed from M. pygmaeusin digestive patterns generated by AseI, XbaI, and MseI on 16S rRNA and in RAPD profiles produced by the primers OPA-18 and OPA-20. These results demonstrate that on Dittrichia plants there is a distinct dimorphic taxon, M. melanotoma, as it is the only species of the genus Macrolophus bearing a first antennal segment with a central white band. Given the limitation of the coloration pattern, the mtDNA genetic markers are the appropriate method for the identification of M. melanotomaand M. pygmaeus.     

Macrolophus pygmaeus (Hemiptera: Miridae) population parameters and biological characteristics when feeding on eggplant and tomato without prey

The population parameters and biological characteristics of the predator Macrolophus pygmaeus Rambur when feeding on eggplant, Solanum melongena L., and tomato, Lycopersicon esculentum (Miller) (both Solanaceae), without access to prey, were investigated at several temperatures. The experiments were conducted at 15, 20, 25, and 30 degrees C, 65 +/- 5% RH, and a photoperiod of 16:8 (L:D) h. Females oviposited on both plant species at all temperatures, with the exception of tomato at 30 degrees C. Fecundity was highest at 20 degrees C (21.55 and 8.28 eggs per female on eggplant and tomato, respectively). Adult longevity on both host plants was greatest at 15 degrees C, reaching 38.72 and 34.20 d for females and 92.88 and 62.80 d for males, on eggplant and tomato, respectively. The estimated values of population parameters showed that this predator increased its numbers on eggplant at all temperatures tested, but the highest intrinsic rate of increase occurred at 25 degrees C (0.0401 d(-1)). On tomato, this predator could not increase in number, although it could survive for a relatively long period. Results demonstrated that M. pygmaeus can survive on both host plants in periods of prey scarcity, particularly on eggplant. Therefore, phytophagy can positively contribute to the effectiveness of this biological control agent. The importance of eggplant as a reservoir for the predator in mixed crops that are less suitable host plants is discussed.     

Real-time PCR quantitation of glucocorticoid receptor alpha isoform

Background  

The expression of glucocorticoid-receptor (GR) seems to be a key mechanism in the regulation of glucocorticoid (GC) sensitivity and is potentially involved in cases of GC resistance or hypersensitivity. The aim of this study is to describe a method for quantitation of GR alpha isoform (GRα) expression using real-time PCR (qrt-PCR) with analytical capabilities to monitor patients, offering standard-curve reproducibility as well as intra- and inter-assay precision.     

Consumption rate, functional response and preference of the predaceous mite Iphiseius degenerans to Tetranychus urticae and Eutetranychus orientalis

The functional response of females of the phytoseiid mite, Iphiseius degenerans (Berlese), to increasing densities of females of its prey, Tetranychus urticae Koch and Eutetranychus orientalis Klein, on bean leaves, were studied under laboratory conditions. Our results indicated that the predator consumed significantly more items of E. orientalis than of T. urticae at all densities treatments. Daily consumption of the predator increased with increasing prey density until a plateau was reached-maximum number of prey consumed was ca. 4 for T. urticae and ca. 12 for E. orientalis. A Type II functional response was determined by a logistic regression model. The highest estimated value a (instantaneous rate of attack) and the lowest value of T ( h ) (handling time) were found for the predator feeding on E. orientalis. Prey selection was evaluated by simultaneously presenting both prey species to the predator in various ratios and at increasing densities. I. degenerans showed a higher predation rate and higher preference for E. orientalis at all the ratios and prey densities tested. This may be due to the smaller size or the inactivity of E. orientalis and the inability of the predator to cope with the webbing of T. urticae. Our results suggest that I. degenerans can be considered a suitable biological control candidate based on its preference for E. orientalis in the Mediterranean region.