Oligonucleotide-stabilized fluorescent silver nanoclusters for turn-on detection of melamine

Fluorescence nanoclusters have been used for the determination of melamine for the first time. The method is based on the fluorescence turn-on of oligonucleotide-stabilized silver nanoclusters (DNA-Ag NCs) by melamine. The enhancement factors (I-I(0))/I(0) increase linearly with melamine concentrations over the range 5.0×10(-8)-7.0×10(-6) M (R(2)=0.998). The detection limit is 1.0×10(-8) M, which is approximately 2000 times lower than the US Food and Drug Administration estimated melamine safety limit of 20.0 μM. Furthermore, the milk samples spiked with melamine are analyzed with excellent recoveries.     

Use of flux pre-analysis to enable ¹³C tracer studies in pyruvate kinase-deficient Escherichia coli

Pyruvate kinase-deficient Escherichia coli (PB25) is a low by-product-producing yet fast-growing mutant that has been shown to have technological potential. Determining the flux limits through finding the extreme point flux sets was previously reported to identify alternate metabolite trafficking scenarios. Previously, the extreme point flux sets were used to design tracer experiments; however, variation in extracellular measurements was not considered, and reaction reversibility was assumed to be low to moderate. In this study, we examined the utility of limiting the fluxes and predetermining the trafficking scenarios in PB25, including confirmation of quasi-linearity between extreme points to ensure sensitivity is maintained. The effects of variation in extracellular measurements and reaction reversibilities were also examined. Tightened flux limits reduced the nonlinearity between label distribution and fluxes. For low to moderate reversibility, contrast was also preserved. However, for highly reversible phosphoglucoisomerase activity, information from common analytes could lead to a flux solution that is biased towards one extreme point. Based on the PB25 model, some suggestions are provided for how predetermining flux limits and trafficking scenarios could enable flux identification in larger network problems.     

Nanospray FAIMS fractionation provides significant increases in proteome coverage of unfractionated complex protein digests

High-field asymmetric waveform ion mobility spectrometry (FAIMS) is an atmospheric pressure ion mobility technique that can be used to reduce sample complexity and increase dynamic range in tandem mass spectrometry experiments. FAIMS fractionates ions in the gas-phase according to characteristic differences in mobilities in electric fields of different strengths. Undesired ion species such as solvated clusters and singly charged chemical background ions can be prevented from reaching the mass analyzer, thus decreasing chemical noise. To date, there has been limited success using the commercially available Thermo Fisher FAIMS device with both standard ESI and nanoLC-MS. We have modified a Thermo Fisher electrospray source to accommodate a fused silica pulled tip capillary column for nanospray ionization, which will enable standard laboratories access to FAIMS technology. Our modified source allows easily obtainable stable spray at flow rates of 300 nL/min when coupled with FAIMS. The modified electrospray source allows the use of sheath gas, which provides a fivefold increase in signal obtained when nanoLC is coupled to FAIMS. In this work, nanoLC-FAIMS-MS and nanoLC-MS were compared by analyzing a tryptic digest of a 1:1 mixture of SILAC-labeled haploid and diploid yeast to demonstrate the performance of nanoLC-FAIMS-MS, at different compensation voltages, for post-column fractionation of complex protein digests. The effective dynamic range more than doubled when FAIMS was used. In total, 10,377 unique stripped peptides and 1649 unique proteins with SILAC ratios were identified from the combined nanoLC-FAIMS-MS experiments, compared with 6908 unique stripped peptides and 1003 unique proteins with SILAC ratios identified from the combined nanoLC-MS experiments. This work demonstrates how a commercially available FAIMS device can be combined with nanoLC to improve proteome coverage in shotgun and targeted type proteomics experiments.     

New species of the genus Chelopistes (Ischnocera: Philopteridae) from Pakistan

A new species of Chelopistes Kéler from common turkey fowl Meleagris gallopavo L. from Karachi, Pakistan is described in detail with reference to morphology and genitalia. The new species is also compared with its closest known ally, Chelopistes meleagridis, a common cosmopolitan parasite previously described from common turkey fowl.     

Knowledge,Attitudes and Practices of Contraception among Afghan Refugee Women in Pakistan: A Cross-Sectional Study

Background

During the 1980s, approximately three million people migrated from Afghanistan to Pakistan and sought refuge in several cities including the city of Karachi. After the initial settlement of the refugees, the international organizations transitioned the health care of these refugees to the two local non-profit service agencies in Karachi. One of these agencies subsidized health care to the refugees under their care and the other agency encouraged the refugees under their care to utilize governmental and non-governmental private health resources at the disposal of general public. Our objective was to measure the effect of health subsidy on the uptake of contraception among Afghan refugee women and compare them to the group of Afghan women without such a subsidy.

Methodology/Principal Findings

A randomly selected group of 650 married Afghan women-325 women in each group-participated in a detailed survey regarding the knowledge, attitude and practices of family planning and contraceptive use. 90 percent of the women in the health subsidy group had had heard of family planning, compared to the 45 percent in the non-subsidized group. The use of contraceptives was greater than two-fold in the former versus the latter. Results of logistic regression analysis revealed that the refugee women who had had access to subsidized healthcare were significantly more likely to use the contraceptive methods with advancing age as compared to the women in the non-health subsidy group. The difference remained significant after adjusting for other variables.

Conclusions/Significance

Refugee women who are provided subsidized healthcare are more inclined to use contraceptives. It is therefore important that Afghan refugee women living elsewhere in Pakistan be provided healthcare subsidy, whereby their reproductive health indicators could improve with reduced fertility. We strongly encourage facilities introducing such subsidies to refugees in resource poor settings to assess the impact through similar inquiry.     

Evidence of field-evolved resistance to organophosphates and pyrethroids in Chrysoperla carnea (Neuroptera: Chrysopidae)

The toxicity of some of the most commonly used insecticides in the organophosphate and pyrethroid classes were investigated against different Chrysoperla carnea (Stephens) (Neuroptera: Chrysopidae) populations collected over three consecutive years (2005-2007). The populations were tested using leaf dip bioassays for residual effects and topical applications to measure the response of larvae that would come into direct contact with field application of insecticides. In leaf dip assays, the LC50 (micrograms per milliliter; 120 h) values for chlorpyrifos and profenofos were in the range of 59.3-1,023 and 180.02-1,118 respectively. The LC50 values for lambda-cyhalthrin, alphamethrin, and deltamethrin were 359.08-2,677, 112.9-923.5, and 47.81-407.03, respectively. The toxicity for the above insecticides in topical application was similar to toxicity in leaf dip assays. The susceptibility of a laboratory population, which was locally developed and designated as (Lab-PK), to deltamethrin was comparable with another susceptible laboratory population. Resistance ratios for five field populations were generally low to medium for deltamethrin, but high to very high for chlorpyrifos, profenofos, lambda-cyhalthrin and alphamethrin compared with the Lab-PK population. Our data also suggested that the five field populations had multiple resistance to two classes of insecticides. The populations showed resistance to two organophosphates tested and to lambda-cyhalthrin and alphamethrin; however, resistance to deltamethrin was only found at two locations. This pattern indicates occurrence of two divergent patterns of resistance within pyrethroids. The resistance to the insecticides was stable across 3 yr, suggesting field selection for general fitness had also taken place in various populations of C. carnea. The broad spectrum of resistance and stability of resistance to insecticides in C. carnea in the current study suggested that it could be a prime candidate for mass releases and compatible with most spray programs.     

Cross-resistance and genetics of resistance to indoxacarb in Spodoptera litura (Lepidoptera: Noctuidae)

Bioassays (at generation G1) with a newly collected field population of Spodoptera litura (F.) (Lepidoptera: Noctuidae) from Multan, Pakistan, showed resistance ratios of 15, 23, 37, and 16 for indoxacarb, spinosad, abamectin, and emamectin, respectively, compared with a laboratory susceptible population, Lab-PK. At G1, the field population was selected with indoxacarb by using single pair crosses. For Indoxa-SEL, bioassay at G4 found that selection increased resistance ratio to 95 for indoxacarb compared with Lab-PK. Selection with indoxacarb significantly increased resistance to spinosad and emamectin; however, resistance to abamectin was observed to drop. A significant reduction in the resistance to indoxacarb was observed in Indoxa-SEL at G9, indicating unstable resistance to indoxacarb; however, it was stable for fipronil. Synergism tests with microsomal oxidase and esterase-specific inhibitors suggested that the indoxacarb resistance was associated with microsomal oxidase. Reciprocal genetic crosses between Indoxa-SEL and Lab-PK populations indicated that resistance was autosomal and incompletely dominant. Tests of monogenic inheritance suggested that resistance to indoxacarb was controlled by more than one locus.     

Inhalation of sphingosine kinase inhibitor attenuates airway inflammation in asthmatic mouse model

Sphingosine 1-phosphate (S1P) produced by sphingosine kinase (SPHK) is implicated in acute immunoresponses, however, mechanisms of SPHK/S1P signaling in the pathogenesis of bronchial asthma are poorly understood. In this study, we hypothesized that SPHK inhibition could ameliorate lung inflammation in ovalbumin (OVA)-challenged mouse lungs. Six- to eight-week-old C57BL/6J mice were sensitized and exposed to OVA for 3 consecutive days. Twenty-four hours later, mice lungs and bronchoalveolar lavage (BAL) fluid were analyzed. For an inhibitory effect, either of the two different SPHK inhibitors, N,N-dimethylsphingosine (DMS) or SPHK inhibitor [SK-I; 2-(p-hydroxyanilino)-4-(p-chlorophenyl) thiazole], was nebulized for 30 min before OVA inhalation. OVA inhalation caused S1P release into BAL fluid and high expression of SPHK1 around bronchial epithelial walls and inflammatory areas. DMS or SK-I inhalation resulted in a decrease in S1P amounts in BAL fluid to basal levels, accompanied by decreased eosinophil infiltration and peroxidase activity. The extent of inhibition caused by DMS inhalation was higher than that caused by SK-I. Like T helper 2 (Th2) cytokine release, OVA inhalation-induced increase in eotaxin expression was significantly suppressed by DMS pretreatment both at protein level in BAL fluid and at mRNA level in lung homogenates. Moreover, bronchial hyperresponsiveness to inhaled methacholine and goblet cell hyperplasia were improved by SPHK inhibitors. These data suggest that the inhibition of SPHK affected acute eosinophilic inflammation induced in antigen-challenged mouse model and that targeting SPHK may provide a novel therapeutic tool to treat bronchial asthma.     

Production and characterization of pharmacologically active recombinant human phosphodiesterase 4B in Dictyostelium discoideum

Phosphodiesterase 4B (PDE4B) is an important therapeutic target for asthma and chronic obstructive pulmonary disease. To identify PDE4 subtype-specific compounds using high-throughput assays, full-length recombinant PDE4 proteins are needed in bulk quantity. In the present study, full-length human PDE4B2 was expressed in the cellular slime mould Dictyostelium discoideum (Dd). A cell density of 2 x 10(7) cells/mL was obtained and up to 1 mg/L recombinant PDE4B2 was purified through Ni-NTA affinity chromatography. The expressed protein was soluble and its activity was comparable to PDE4B2 protein expressed in mammalian cells (K(m)=1.7 microM). The functional significance of the Dd expression system is supported by the demonstration that, in concert with proteins expressed in mammalian systems, there are no major changes in the affinity for PDE4B2 inhibitors and substrates. These findings thus provide the first evidence that Dd can be utilized for the expression and purification of functionally active full-length human PDE4B2 in large amounts required for high-throughput screening of pharmacologically active compounds against this therapeutic target.     

Function and structure of lipid storage droplet protein 1 studied in lipoprotein complexes

Triglycerides (TG) stored in lipid droplets (LDs) are the main energy reserve in all animals. The mechanism by which animals mobilize TG is complex and not fully understood. Several proteins surrounding the LDs have been implicated in TG homeostasis such as mammalian perilipin A and insect lipid storage proteins (Lsd). Most of the knowledge on LD-associated proteins comes from studies using cells or LDs leaving biochemical properties of these proteins uncharacterized. Here we describe the purification of recombinant Lsd1 and its reconstitution with lipids to form lipoprotein complexes suitable for functional and structural studies. Lsd1 in the lipid bound state is a predominately α-helical protein. Using lipoprotein complexes containing triolein it is shown that PKA mediated phosphorylation of Lsd1 promoted a 1.7-fold activation of the main fat body lipase demonstrating the direct link between Lsd1 phosphorylation and activation of lipolysis. Serine 20 was identified as the Lsd1-phosphorylation site triggering this effect.