Assessing the reliability of eBURST using simulated populations with known ancestry

Background  

The program eBURST uses multilocus sequence typing data to divide bacterial populations into groups of closely related strains (clonal complexes), predicts the founding genotype of each group, and displays the patterns of recent evolutionary descent of all other strains in the group from the founder. The reliability of eBURST was evaluated using populations simulated with different levels of recombination in which the ancestry of all strains was known.     

PheMaDB: A solution for storage,retrieval, and analysis of high throughput phenotype data

Background  

OmniLog™ phenotype microarrays (PMs) have the capability to measure and compare the growth responses of biological samples upon exposure to hundreds of growth conditions such as different metabolites and antibiotics over a time course of hours to days. In order to manage the large amount of data produced from the OmniLog™ instrument, PheMaDB (Phenotype Microarray DataBase), a web-based relational database, was designed. PheMaDB enables efficient storage, retrieval and rapid analysis of the OmniLog™ PM data.     

Association of mast cells with lung function in chronic obstructive pulmonary disease

Background

Surfactant protein D (SP-D), an innate immune molecule, plays an important protective role during airway inflammation. Deficiency of this molecule induces emphysematous changes in murine lungs, but its significance in human COPD remains unclear.

Methods

We collected bronchoalveolar lavage fluid from 20 subjects with varying degrees of COPD (8 former smokers and 12 current smokers) and 15 asymptomatic healthy control subjects (5 never smokers, 3 remote former smokers, and 7 current smokers). All subjects underwent a complete medical history and pulmonary function testing. SP-D was measured by Enzyme-Linked ImmunoSorbent Assay. Statistical analysis was performed using nonparametric methods and multivariable linear regression for control of confounding. The effect of corticosteroid treatment on SP-D synthesis was studied in vitro using an established model of isolated type II alveolar epithelial cell culture.

Results

Among former smokers, those with COPD had significantly lower SP-D levels than healthy subjects (median 502 and 1067 ng/mL, respectively, p = 0.01). In a multivariable linear regression model controlling for age, sex, race, and pack-years of tobacco, COPD was independently associated with lower SP-D levels (model coefficient -539, p = 0.04) and inhaled corticosteroid use was independently associated with higher SP-D levels (398, p = 0.046). To support the hypothesis that corticosteroids increase SP-D production we used type II alveolar epithelial cells isolated from adult rat lungs. These cells responded to dexamethasone treatment by a significant increase of SP-D mRNA (p = 0.041) and protein (p = 0.037) production after 4 days of culture.

Conclusion

Among former smokers, COPD is associated with lower levels of SP-D and inhaled corticosteroid use is associated with higher levels of SP-D in the lung. Dexamethasone induced SP-D mRNA and protein expression in isolated epithelial cells in vitro. Given the importance of this molecule as a modulator of innate immunity and inflammation in the lung, low levels may play a role in the pathogenesis and/or progression of COPD. Further, we speculate that inhaled steroids may induce SP-D expression and that this mechanism may contribute to their beneficial effects in COPD. Larger, prospective studies are warranted to further elucidate the role of surfactant protein D in modulating pulmonary inflammation and COPD pathogenesis.     

Functional changes in human leukemic cell line HL-60. A model for myeloid differentiation

Polar solvents induce terminal differentiation in the human promyelocytic leukemia cell line HL-60. The present studies describe the functional changes that accompany the morphologic progression from promyelocytes to bands and poly-morphonuclear leukocytes (PMN) over 9 d of culture in 1.3 percent dimethylsulfoxide (DMSO). As the HL-60 cells mature, the rate of O(2-) production increase 18-fold, with a progressive shortening of the lag time required for activation. Hexosemonophosphate shunt activity rises concomitantly. Ingestin of paraffin oil droplets opsonized with complement or Ig increases 10-fold over 9 d in DMSO. Latex ingestion per cell by each morphologic type does not change significantly, but total latex ingestion by groups of cells increases with the rise in the proportion of mature cells with greater ingestion capacities. Degranulation, as measured by release of β-glucuronidase, lysozyme, and peroxidase, reaches maximum after 3-6 d in DMSO, then declines. HL-60 cells contain no detectable lactoferrin, suggesting that their secondary granules are absent or defective. However, they kill staphylococci by day 6 in DMSO. Morphologically immature cells (days 1-3 in DMSO) are capable of O(2-) generation, hexosemonophosphate shunt activity, ingestion, degranulation, and bacterial killing. Maximal performance of each function by cells incubated in DMSO for longer periods of time is 50-100 percent that of normal PMN. DMSO- induced differentiation of HL-60 cells is a promising model for myeloid development.     

Characterization and PCR multiplexing of polymorphic microsatellite loci for the invasive ant Wasmannia auropunctata

Highly polymorphic genetic markers provide a useful tool for estimating genetic parameters in studies of the evolution of sociality in insects. We isolated and characterized 12 polymorphic microsatellite loci in the invasive ant, Wasmannia auropunctata, and described experimental conditions for PCR (polymerase chain reaction) multiplexing and simultaneously genotyping these loci in two sets of five and seven markers. The number of alleles per locus ranged from two to 14 and the observed heterozygosity ranged from 0.233 to 0.967. Moreover, results of cross‐species amplification tests are reported in three other species of Wasmannia and in two species of the genus Allomerus.     

Development, Optimization, and Characterization of Solid Self-Nanoemulsifying Drug Delivery Systems of Valsartan Using Porous Carriers

The present studies entail formulation development of novel solid self-nanoemulsifying drug delivery systems (S-SNEDDS) of valsartan with improved oral bioavailability, and evaluation of their in vitro and in vivo performance. Preliminary solubility studies were carried out and pseudoternary phase diagrams were constructed using blends of oil (Capmul MCM), surfactant (Labrasol), and cosurfactant (Tween 20). The SNEDDS were systematically optimized by response surface methodology employing 3^3-Box–Behnken design. The prepared SNEDDS were characterized for viscocity, refractive index, globule size, zeta potential, and TEM. Optimized liquid SNEDDS were formulated into free flowing granules by adsorption on the porous carriers like Aerosil 200, Sylysia (350, 550, and 730) and Neusilin US2, and compressed into tablets. In vitro dissolution studies of S-SNEDDS revealed 3–3.5-fold increased in dissolution rate of the drug due to enhanced solubility. In vivo pharmacodynamic studies in Wistar rats showed significant reduction in mean systolic BP by S-SNEDDS vis-à-vis oral suspension (p < 0.05) owing to the drug absorption through lymphatic pathways. Solid-state characterization of S-SNEDDS using FT-IR and powder XRD studies confirmed lack of any significant interaction of drug with lipidic excipients and porous carriers. Further, the accelerated stability studies for 6 months revealed that S-SNEDDS are found to be stable without any change in physiochemical properties. Thus, the present studies demonstrated the bioavailability enhancement potential of porous carriers based S-SNEDDS for a BCS class II drug, valsartan.KEY WORDS: BCS, bioavailability, in vitro dissolution, porous carriers, XRD     

Evaporation and carbonic anhydrase activity recorded in oxygen isotope signatures of net CO2 fluxes from a Mediterranean soil

The oxygen stable isotope composition (δ¹⁸O) of CO₂ is a valuable tool for studying the gas exchange between terrestrial ecosystems and the atmosphere. In the soil, it records the isotopic signal of water pools subjected to precipitation and evaporation events. The δ¹⁸O of the surface soil net CO₂ flux is dominated by the physical processes of diffusion of CO₂ into and out of the soil and the chemical reactions during CO₂–H₂O equilibration. Catalytic reactions by the enzyme carbonic anhydrase, reducing CO₂ hydration times, have been proposed recently to explain field observations of the δ¹⁸O signatures of net soil CO₂ fluxes. How important these catalytic reactions are for accurately predicting large‐scale biosphere fluxes and partitioning net ecosystem fluxes is currently uncertain because of the lack of field data. In this study, we determined the δ¹⁸O signatures of net soil CO₂ fluxes from soil chamber measurements in a Mediterranean forest. Over the 3 days of measurements, the observed δ¹⁸O signatures of net soil CO₂ fluxes became progressively enriched with a well‐characterized diurnal cycle. Model simulations indicated that the δ¹⁸O signatures recorded the interplay of two effects: (1) progressive enrichment of water in the upper soil by evaporation, and (2) catalytic acceleration of the isotopic exchange between CO₂ and soil water, amplifying the contributions of ‘atmospheric invasion’ to net signatures. We conclude that there is a need for better understanding of the role of enzymatic reactions, and hence soil biology, in determining the contributions of soil fluxes to oxygen isotope signals in atmospheric CO₂.     

Non-infectious aggregates of the prion protein react with several PrPSc-directed antibodies

The key event in the pathogenesis of prion diseases is the conformational conversion of the normal prion protein (PrP) (PrP^C) into an infectious, aggregated isoform (PrP^Sc) that has a high content of β-sheet. Historically, a great deal of effort has been devoted to developing antibodies that specifically recognize PrP^Sc but not PrP^C, as such antibodies would have enormous diagnostic and experimental value. A mouse monoclonal IgM antibody (designated 15B3) and three PrP motif-grafted monoclonal antibodies (referred to as IgG 19–33, 89–112, and 136–158) have been previously reported to react specifically with infectious PrP^Sc but not PrP^C. In this study, we extend the characterization of these four antibodies by testing their ability to immunoprecipitate and immunostain infectious and non-infectious aggregates of wild-type, mutant, and recombinant PrP. We find that 15B3 as well as the motif-grafted antibodies recognize multiple types of aggregated PrP, both infectious and non-infectious, including forms found in brain, in transfected cells, and induced in vitro from purified recombinant protein. These antibodies are exquisitely selective for aggregated PrP, and do not react with soluble PrP even when present in vast excess. Our results suggest that 15B3 and the motif-grafted antibodies recognize structural features common to both infectious and non-infectious aggregates of PrP. Our study extends the utility of these antibodies for diagnostic and experimental purposes, and it provides new insight into the structural changes that accompany PrP oligomerization and prion propagation.