Two independent replicons can support replication of the anthrax toxin-encoding plasmid pXO1 of Bacillus anthracis

The large pXO1 plasmid (181.6kb) of Bacillus anthracis encodes the anthrax toxin proteins. Previous studies have shown that two separate regions of pXO1 can support replication of pXO1 miniplasmids when introduced into plasmid-less strains of this organism. No information is currently available on the ability of the above two replicons, termed RepX and ORFs 14/16 replicons, to support replication of the full-length pXO1 plasmid. We generated mutants of the full-length pXO1 plasmid in which either the RepX or the ORFs 14/16 replicon was inactivated by TargeTron insertional mutagenesis. Plasmid pXO1 derivatives containing only the RepX or the ORFs 14/16 replicon were able to replicate when introduced into a plasmid-less B. anthracis strain. Plasmid copy number analysis showed that the ORFs 14/16 replicon is more efficient than the RepX replicon. Our studies demonstrate that both the RepX and ORFs 14/16 replicons can independently support the replication of the full-length pXO1 plasmid.     

Unusual polyphosphate inclusions observed in a marine Beggiatoa strain

Sulfide-oxidizing bacteria of the genus Beggiatoa are known to accumulate phosphate intracellularly as polyphosphate but little is known about the structure and properties of these inclusions. Application of different staining techniques revealed the presence of unusually large polyphosphate inclusions in the marine Beggiatoa strain 35Flor. The inclusions showed a co-occurrence of polyphosphate, calcium and magnesium when analyzed by scanning electron microscopy and energy dispersive X-ray analysis. Similar to polyphosphate-enriched acidocalcisomes of prokaryotes and eukaryotes, the polyphosphate inclusions in Beggiatoa strain 35Flor are enclosed by a lipid layer and store cations. However, they are not notably acidic. 16S rRNA gene sequence-based phylogenetic reconstruction showed an affiliation of Beggiatoa strain 35Flor to a monophyletic branch, comprising other narrow vacuolated and non-vacuolated Beggiatoa species. The polyphosphate inclusions represent a new type of membrane surrounded storage compartment within the genus Beggiatoa, distinct from the mostly nitrate-storing vacuoles known from other marine sulfide-oxidizing bacteria of the family Beggiatoaceae.     

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.     

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.     

Molecular Epidemiology of Influenza A(H1N1)pdm09 Viruses from Pakistan in 2009-2010

Background

In early 2009, a novel influenza A(H1N1) virus that emerged in Mexico and United States rapidly disseminated worldwide. The spread of this virus caused considerable morbidity with over 18000 recorded deaths. The new virus was found to be a reassortant containing gene segments from human, avian and swine influenza viruses.

Methods/Results

The first case of human infection with A(H1N1)pdm09 in Pakistan was detected on 18^th June 2009. Since then, 262 laboratory-confirmed cases have been detected during various outbreaks with 29 deaths (as of 31^st August 2010). The peak of the epidemic was observed in December with over 51% of total respiratory cases positive for influenza. Representative isolates from Pakistan viruses were sequenced and analyzed antigenically. Sequence analysis of genes coding for surface glycoproteins HA and NA showed high degree of high levels of sequence identity with corresponding genes of regional viruses circulating South East Asia. All tested viruses were sensitive to Oseltamivir in the Neuraminidase Inhibition assays.

Conclusions

Influenza A(H1N1)pdm09 viruses from Pakistan form a homogenous group of viruses. Their HA genes belong to clade 7 and show antigenic profile similar to the vaccine strain A/California/07/2009. These isolates do not show any amino acid changes indicative of high pathogenicity and virulence. It is imperative to continue monitoring of these viruses for identification of potential variants of high virulence or drug resistance.     

Mir-206 Regulates Pulmonary Artery Smooth Muscle Cell Proliferation and Differentiation

Pulmonary Arterial Hypertension (PAH) is a progressive devastating disease characterized by excessive proliferation of the Pulmonary Arterial Smooth Muscle Cells (PASMCs). Studies suggest that PAH and cancers share an apoptosis-resistant state featuring excessive cell proliferation. MicroRNA-206 (miR-206) is known to regulate proliferation and is implicated in various types of cancers. However, the role of miR-206 in PAH has not been studied. In this study, it is hypothesized that miR-206 could play a role in the proliferation of PASMCs. In the present study, the expression patterns of miR-206 were investigated in normal and hypertensive mouse PASMCs. The effects of miR-206 in modulating cell proliferation, apoptosis and smooth muscle cell markers in human pulmonary artery smooth muscle cells (hPASMCs) were investigated in vitro. miR-206 expression in mouse PASMCs was correlated with an increase in right ventricular systolic pressure. Reduction of miR-206 levels in hPASMCs causes increased proliferation and reduced apoptosis and these effects were reversed by the overexpression of miR-206. miR-206 over expression also increased the levels of smooth muscle cell differentiation markers α-smooth muscle actin and calponin implicating its importance in the differentiation of SMCs. miR-206 overexpression down regulated Notch-3 expression, which is key a factor in PAH development. These results suggest that miR-206 is a potential regulator of proliferation, apoptosis and differentiation of PASMCs, and that it could be used as a novel treatment strategy in PAH.