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.     

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.     

Substituting dietary linoleic acid with α-linolenic acid improves insulin sensitivity in sucrose fed rats

This study describes the effect of substituting dietary linoleic acid (18:2 n-6) with α-linolenic acid (18:3 n-3) on sucrose-induced insulin resistance (IR). Wistar NIN male weanling rats were fed casein based diet containing 22 energy percent (en%) fat with ～6, 9 and 7 en% saturated fatty acids (SFA), monounsaturated fatty acids (MUFA) and polyunsaturated fatty acids (PUFA) respectively for 3 months. IR was induced by replacing starch (ST) with sucrose (SU). Blends of groundnut, palmolein, and linseed oil in different proportions furnished the following levels of 18:3 n-3 (g/100 g diet) and 18:2 n-6/18:3 n-3 ratios respectively: ST-220 (0.014, 220), SU-220 (0.014, 220), SU-50 (0.06, 50), SU-10 (0.27, 10) and SU-2 (1.1, 2). The results showed IR in the sucrose fed group (SU-220) as evidenced by increase in fasting plasma insulin and area under the curve (AUC) of insulin in response to oral glucose load. In SU-220, the increase in adipocyte plasma membrane cholesterol/phospholipid ratio was associated with a decrease in fluidity, insulin stimulated glucose transport, antilipolytic effect of insulin and increase in basal and norepinephrine stimulated lipolysis in adipocytes. In SU-50, sucrose induced alterations in adipocyte lipolysis and antilipolysis were normalized. However, in SU-2, partial corrections in plasma insulin, AUC of insulin and adipocyte insulin stimulated glucose transport were observed. Further, plasma triglycerides and cholesterol decreased in SU-2. In diaphragm phospholipids, the observed dose dependent increase in long chain (LC) n-3 PUFA was associated with a decrease in LC-n-6 PUFA but insulin stimulated glucose transport increased only in SU-2. Thus, this study shows that the substitution of one-third of dietary 18:2 n-6 with 18:3 n-3 (SU-2) results in lowered blood lipid levels and increases peripheral insulin sensitivity, possibly due to the resulting high LCn-3 PUFA levels in target tissues of insulin action. These findings suggest a role for 18:3 n-3 in the prevention of insulin resistant states. The current recommendation to increase 18:3 n-3 intake for reducing cardiovascular risk may also be beneficial for preventing IR in humans.     

Culture-Based Strategies for Reduction of Protease Activity in Filtrates from Aspergillus niger NRRL-3

Summary While Aspergillus strains are also being considered as potential hosts for production of extracellular heterologous proteins, the proteases produced by the host are highly problematic in that they typically modify and degrade the recombinant proteins. Culture-based approaches for minimization of protease activity in culture supernatants of Aspergillus niger NRRL-3 included reduction or elimination of peptide nitrogen in the medium, preferential use of a defined salts medium rather than a non-peptide nitrogen medium containing yeast-nitrogen base, supplementation of the medium with carboxymethylcellulose and cultivation at pH 6.5 rather than 7.5. In general, increased proteolytic activity was observed after maximum biomass was observed and biomass was declining suggesting the majority of protease activity was released by cell lysis. Carboxymethylcellulose shifted mycelial morphology from pelleted to filamentous. Mycelium lysis in the centre of pellets, with resultant release of intracellular proteases, would explain why filamentous cultures exhibited much lower proteolytic activity than pelleted cultures.     

Calcium Ions as “Miscibility Switch”: Colocalization of Surfactant Protein B with Anionic Lipids under Absolute Calcium Free Conditions

One of the main determinants of lung surfactant function is the complex interplay between its protein and lipid components. The lipid specificity of surfactant protein B (SP-B), however, and the protein's ability to selectively squeeze out lipids, has remained contradictory. In this work we present, for the first time to our knowledge, by means of time-of-flight secondary ion mass spectrometry chemical imaging, a direct evidence for colocalization of SP-B as well as its model peptide KL₄ with negatively charged dipalmitoylphosphatidylglycerol under absolute calcium free conditions. Our results prove that protein/lipid localization depends on the miscibility of all surfactant components, which itself is influenced by subphase ionic conditions. In contrast to our earlier studies reporting SP-B/KL₄ colocalization with zwitterionic dipalmitoylphosphatidylcholine, in the presence of even the smallest traces of calcium, we finally evidence an apparent reversal of protein/lipid mixing behavior upon calcium removal with ethylene diamine tetraacetic acid. In addition, scanning force microscopy measurements reveal that by depleting the subphase from calcium ions the protrusion formation ability of SP-B or KL₄ is not hampered. However, in the case of KL₄, distinct differences in protrusion morphology and height are visible. Our results support the idea that calcium ions act as a “miscibility switch” in surfactant model systems and probably are one of the major factors steering lipid/protein mixing behavior as well as influencing the protein's protrusion formation ability.