Seasonal variations in the mating-related traits of Drosophila melanogaster

The Indian subcontinent shows high levels of seasonal weather variation, but the extent to which mating-related traits (mating latency, copulation duration and number of progeny produced) are being affected by such variations in Drosophila species remain poorly understood. In the present study, we analyzed the effects of seasonal change (humidity and temperature) on mating-related traits of Drosophila melanogaster by mimicking natural conditions in the laboratory. The light body color phenotype is collected in large numbers during the rainy season, while the dark phenotype is prevalent in the winter. We found that a short-term stress, in the form of reduced humidity or temperature, causes a strong climatic selection pressure, which leads to assortative mating and longer copulation duration of the dark phenotype. By contrast, the light phenotype shows higher assortative mating and longer copulation duration after short-term high humidity or high temperature stress. Higher assortative mating and increased copulation duration results in high progeny numbers which may be a cause for the high prevalence of the dark phenotype in winter and the light phenotype in the rainy season. Thus, besides plasticity, seasonal changes in mating propensity can be a potential cause of the change in the frequency of the dark and light phenotypes of D. melanogaster during different seasons.     

IgE production in CD40/CD40L cross-talk of B and mast cells and mediator release via TGase 2 in mouse allergic asthma

TGase 2 is over-expressed in a variety of inflammatory diseases including allergic asthma. This study aimed to investigate the role of TGase 2 on IgE production and signaling pathways in mast cell activation related to OVA-induced allergic asthma. Bone marrow-derived mast cells (BMMCs) isolated from WT or TGase 2^?/? mice were activated with Ag/Ab (refer to act-WT-BMMCs and act-KO-BMMCs, respectively). B cells isolated from splenocytes were activated with anti-mouse IgM (act-B cells), and B cells were co-cultured with BMMCs. WT and TGase 2^?/? mice were sensitized and challenged with OVA adsorbed in alum hydroxide. Intracellular Ca^2 + ([Ca^2 +]_i) levels were determined by fluorescence intensity; IgE, mediators and TGase 2 activity by ELISA; the CD138 expression by FACS analyzer; cell surface markers and signal molecules by Western blot; NF-κB by EMSA; co-localization of mast cells and B cells by immunohistochemistry; Fcε RI-mediated mast cell activation by PCA test; expression of cytokines, MMPs, TIMPs, TLR2 and Fc?RI by RT-PCR. In vitro, act-KO-BMMCs reduced the [Ca^2 +]i levels, NF-κB activity, expression of CD40/CD40L, plasma cells, total IgE levels and TGase 2 activity in act-B cells co-cultured with act-BMMCs, expression of inflammatory cytokines and MMPs2/9, release of mediators (TNF-α, LTs and cytokines), and activities of signal molecules (PKCs, MAP kinases, I-κB and PLA2), which were all increased in act-WT-BMMCs. TGase 2 siRNA transfected/activated-BMMCs reduced all responses as same as those in act-KO-BMMCs. In allergic asthma model, TGase 2^?/? mice protected against PCA reaction, OVA-specific IgE production and AHR, and they reduced co-localization of mast cells and B cells or IgE in lung tissues, expression and co-localization of surface molecules in mast cells (c-kit and CD40L) and B cells (CD23 and CD40), inflammatory cells including mast cells, goblet cells, amounts of collagen and mediator release in BAL fluid and/or lung tissues, which were all increased in WT mice. TLR expression in TGase 2^?/? mice did not differ from those in WT mice. Our data suggest that TGase 2 expression and Ca^2 + influx required by bidirectional events in mast cell activation facilitate IgE production in B cells via up-regulating mast cell CD40L expression, and induce the expression of numerous signaling molecules associated with airway inflammation and remodeling in allergic asthma.     

The Limitations of an Exclusively Colloidal View of Protein Solution Hydrodynamics and Rheology

Proteins are complex macromolecules with dynamic conformations. They are charged like colloids, but unlike colloids, charge is heterogeneously distributed on their surfaces. Here we overturn entrenched doctrine that uncritically treats bovine serum albumin (BSA) as a colloidal hard sphere by elucidating the complex pH and surface hydration-dependence of solution viscosity. We measure the infinite shear viscosity of buffered BSA solutions in a parameter space chosen to tune competing long-range repulsions and short-range attractions (2 mg/mL ≤ [BSA] ≤ 500 mg/mL and 3.0 ≤ pH ≤ 7.4). We account for surface hydration through partial specific volume to define volume fraction and determine that the pH-dependent BSA intrinsic viscosity never equals the classical hard sphere result (2.5). We attempt to fit our data to the colloidal rheology models of Russel, Saville, and Schowalter (RSS) and Krieger-Dougherty (KD), which are each routinely and successfully applied to uniformly charged suspensions and to hard-sphere suspensions, respectively. We discover that the RSS model accurately describes our data at pH 3.0, 4.0, and 5.0, but fails at pH 6.0 and 7.4, due to steeply rising solution viscosity at high concentration. When we implement the KD model with the maximum packing volume fraction as the sole floating parameter while holding the intrinsic viscosity constant, we conclude that the model only succeeds at pH 6.0 and 7.4. These findings lead us to define a minimal framework for models of crowded protein solution viscosity wherein critical protein-specific attributes (namely, conformation, surface hydration, and surface charge distribution) are addressed.     

Identification of specific IgE binding proteins in Castor bean (Ricinus communis) pollen obtained from different source materials

Allergenic components of Ricinus communis pollen obtained from different stages of inflorescence, different time intervals, different years and places were studied by immunoblot analysis. Proteins separated by SDS-PAGE and transferred to NC were identified using pooled sera from 15 skin and RAST positive patients. The IgE binding components in M.W. range of 14 to 70 kD were identified. The protein fractions of 70, 66, 64, 60, 50, 45, 36, 22 and 14 kD are the most prominent allergenic bands. Six samples collected during same pollination season from the same place showed similar allergenic profile. Of the samples collected from different stages of inflorescence, pollen of immature buds showed only three bands as compared to 18 from mature buds and flowers. Variability was seen in the IgE binding components of pollen stored for different years and obtained from different geographic regions of India. The IgE binding pattern of fifteen sera were heterogenous. The number of bands identified by different sera varied from 3 to 18. Two protein components of 66 and 36 kD were recognised by 14 (93.3%) of the 15 sera studied. The result suggests that there exists variations in the specific IgE binding pattern in pollen samples of Castor Bean, obtained from difference source materials.     

A novel technique for loading of paclitaxel-PLGA nanoparticles onto ePTFE vascular grafts

The major cause of hemodialysis vascular access dysfunction (HVAD) is the occurrence of stenosis followed by thrombosis at venous anastomosis sites due to the aggressive development of venous neointimal hyperplasia. Local delivery of antiproliferative drugs may be effective in inhibiting hyperplasia without causing systemic side effects. We have previously demonstrated that paclitaxel-coated expanded poly(tetrafluoroethylene) (ePTFE) grafts, by a dipping method, could prevent neointimal hyperplasia and stenosis of arteriovenous (AV) hemodialysis grafts, especially at the graft-venous anastomoses; however, large quntities of initial burst release have remained a problem. To achieve controlled drug release, paclitaxel (Ptx)-loaded poly(lactic-co-glycolic acid) (PLGA) nanoparticles (Ptx-PLGA-NPs) were prepared by the emulsion-solvent evaporation method and then transferred to the luminal surface and inner part of ePTFE vascular grafts through our micro tube pumping and spin penetration techniques. Scanning electron microscope (SEM) images of various stages of Ptx-PLGA-NPs unequivocally showed that micro tube pumping followed by spin penetration effectively transferred Ptx-PLGA-NPs to the inner part, as well as the luminal surface, of an ePTFE graft. In addition, the in vitro release profiles of paclitaxel demonstrated that this new system achieved controlled drug delivery with a reduced initial burst release. These results suggest that loading of Ptx-PLGA-NPs to the luminal surface and the inner part of an ePTFE graft is a promising strategy to ultimately inhibit the development of venous neointimal hyperplasia.     

An aptasensor for rapid and sensitive detection of estrogen receptor alpha in human breast cancer

The analysis of estrogen receptor (ER) expression in breast carcinomas plays a crucial role in determining the endocrine responsiveness of tumors for systemic adjuvant therapy. Conventionally, the ER levels in breast carcinomas had been detected using the dextran-coated charcoal assay and radioimmunoassay, which are now substituted with safer and economic antibody-based assays such as immunohistochemistry (IHC) and enzyme-linked immunosorbent assay (ELISA). Despite a gold (Au) standard method, the IHC has been criticized for factors such as tissue fixation, antibody selection, and threshold staining for result interpretation that could falsify test accuracy and reproducibility. The quest for alternative methods of ER quantification in tissue samples paved the way for aptamer-based diagnostics. Previously, we have isolated a DNA aptamer against human ER alpha (ERα) using an in vitro evolution system. In this study, we developed an electrochemical sensor using the 76-nucleotide DNA ERα- aptamer for rapid, precise, and cost-effective detection of ERα expression in human breast cancer patients. The aptasensor was constructed by covalently immobilizing the thiolated ERα- aptamer onto a screen-printed Au electrode. Construction of aptasensors was confirmed through atomic force microscopy and differential pulse voltammetry measurements. A detection limit of 0.001 ng/ml was calculated for full-length ERα (66.2 kDa) in a detection time of 10 min. Analysis of the cancerous breast tissue samples using the ELISA and aptasensor methods enabled distinctive classification of samples into the categories of ER −ve, weak ER +ve, and strong ER +ve samples. The current change of this aptasensor lies within 5% after a storage of 60 days at 4°C. Further studies on a reasonably large sample size are required to realize the clinical potential of the sensor.     

Effect of anti-mycobacterial antibodies on activation of the alternative pathway of the human complement system

Abstract Genome analysis of Pseudomonas aeruginosa was performed by digestion with rare-cutting restriction endonucleases and subsequent one- and two-dimensional field inversion gel electrophoresis (FIGE). The frequency of chromosomal recognition sites increased in the order Spe I, Dra I, Xba I, Ssp I, Nhe I. The genome size of strain PAO and the 17 IATS strains varied from 4.4 × 10⁶ to 5.4 × 10⁶ base pairs. Double restriction digests and two-dimensional FIGE provide a genome fingerprint which is useful for the identification and typing of the respective strains.     

State of pregnancy modifies lead toxicity in mice

Toxicity of lead acetate after administration through the oral route at 0-50 mg/kg body weight of animal has been assessed in the liver of pregnant mice and compared with the effect in the liver of nonpregnant dams. Analysis showed that the basal level of hepatic lead is considerably reduced during pregnancy as compared to that in nonpregnant state. After administration of Pb-acetate, deposited lead in liver of nonpregnant mice was 3- to 4-fold while in pregnant mice was, it was 1.8- to 3.0-fold over their respective control values. Although hepatic Fe, Cu, and Zn levels had a tendency to be lowered during pregnancy, it appeared that the added trace quantity of lead prior to and during pregnancy helped in the retention of these metals, which either remained unaffected (as Fe) or declined (Cu and Zn) after lead administration during the nonpregnant state. The effect of lead on Mn diminution, however, was visible at the dose of 50 mg/kg body wt of lead-acetate. Alkaline phosphatase, which increased during pregnancy along with Mn, was reversed between the pregnant and nonpregnant states after oral administration of lead. On the other hand, the level of delta-aminolevolunic acid dehydratase, which declined during normal pregnancy, continued to fall further after lead exposure. It is concluded that the distribution of basal or administered lead and its effect on enzyme activities and trace metal composition in liver depends on the pregnant and nonpregnant states of female hosts.     

Overexpression of phytochelatin synthase in Arabidopsis leads to enhanced arsenic tolerance and cadmium hypersensitivity

Phytochelatin synthase (PCS) catalyzes the final step in the biosynthesis of phytochelatins, which are a family of cysteine-rich thiol-reactive peptides believed to play important roles in processing many thiol-reactive toxicants. A modified Arabidopsis thaliana PCS sequence (AtPCS1) was active in Escherichia coli. When AtPCS1 was overexpressed in Arabidopsis from a strong constitutive Arabidopsis actin regulatory sequence (A2), the A2::AtPCS1 plants were highly resistant to arsenic, accumulating 20-100 times more biomass on 250 and 300 microM arsenate than wild type (WT); however, they were hypersensitive to Cd(II). After exposure to cadmium and arsenic, the overall accumulation of thiol-peptides increased to 10-fold higher levels in the A2::AtPCS1 plants compared with WT, as determined by fluorescent HPLC. Whereas cadmium induced greater increases in traditional PCs (PC2, PC3, PC4), arsenic exposure resulted in the expression of many unknown thiol products. Unexpectedly, after arsenate or cadmium exposure, levels of the dipeptide substrate for PC synthesis, gamma-glutamyl cysteine (gamma-EC), were also dramatically increased. Despite these high thiol-peptide concentrations, there were no significant increases in concentrations of arsenic and cadmium in above-ground tissues in the AtPCS1 plants relative to WT plants. The potential for AtPCS1 overexpression to be useful in strategies for phytoremediating arsenic and to compound the negative effects of cadmium are discussed.     

Nucleic acid capture assay,a new method for direct quantitation of nucleic acids

Technologies allowing direct detection of specific RNA/DNA sequences occasionally serve as an alternative to amplification methods for gene expression studies. In these direct methods the hybridization of probes takes place in complex mixtures, thus specificity and sensitivity still limit the use of current technologies. To address these challenges, we developed a new technique called the nucleic acid capture assay, involving a direct multi-capture system. This approach combines a 3′-ethylene glycol scaffolding with the incorporation of 2′-methoxy deoxyribonucleotides in the capture sequences. In our design, all nucleotides other than those complementary to the target mRNA have been replaced by an inert linker, resulting in significant reductions in non-specific binding. We also provide a versatile method to detect the presence of captured targets by using specific labeled probes with alkaline phosphatase-conjugated anti-label antibodies. This direct, flexible and reliable technique for gene expression analysis is well suited for high-throughput screening and has potential for DNA microarray applications.