In vivo osteopontin-induced macrophage accumulation is dependent on CD44 expression

In order to assess the role of osteopontin (OPN) in leukocyte accumulation in inflammatory conditions, native OPN and its thrombin cleaved form (OPN + Thr) were studied in vivo using a rodent subcutaneous air pouch model (AP). Both forms of OPN-induced macrophage infiltration into the AP in wild-type mice. In animals lacking CD44, macrophage numbers were significantly reduced within the cavity, but cells still accumulated along the subcutaneous lining. In animals lacking endogenous OPN, no differences were found in exogenous OPN-induced macrophage accumulation, although macrophage exhibited increased α4 integrin expression. These studies reveal that both OPN and OPN + Thr attract macrophages in vivo through CD44.     

Relative Wulst volume is correlated with orbit orientation and binocular visual field in birds

In mammals, species with more frontally oriented orbits have broader binocular visual fields and relatively larger visual regions in the brain. Here, we test whether a similar pattern of correlated evolution is present in birds. Using both conventional statistics and modern comparative methods, we tested whether the relative size of the Wulst and optic tectum (TeO) were significantly correlated with orbit orientation, binocular visual field width and eye size in birds using a large, multi-species data set. In addition, we tested whether relative Wulst and TeO volumes were correlated with axial length of the eye. The relative size of the Wulst was significantly correlated with orbit orientation and the width of the binocular field such that species with more frontal orbits and broader binocular fields have relatively large Wulst volumes. Relative TeO volume, however, was not significant correlated with either variable. In addition, both relative Wulst and TeO volume were weakly correlated with relative axial length of the eye, but these were not corroborated by independent contrasts. Overall, our results indicate that relative Wulst volume reflects orbit orientation and possibly binocular visual field, but not eye size.     

Cumulative funnel plots for the early detection of interoperator variation: retrospective database analysis of observed versus predicted results of percutaneous coronary intervention

Objective To use funnel plots and cumulative funnel plots to compare in-hospital outcome data for operators undertaking percutaneous coronary interventions with predicted results derived from a validated risk score to allow for early detection of variation in performance.Design Analysis of prospectively collected data.Setting Tertiary centre NHS hospital in the north east of England.Participants Five cardiologists carrying out percutaneous coronary interventions between January 2003 and December 2006.Main outcome measures In-hospital major adverse cardiovascular and cerebrovascular events (in-hospital death, Q wave myocardial infarction, emergency coronary artery bypass graft surgery, and cerebrovascular accident) analysed against the logistic north west quality improvement programme predicted risk, for each operator. Results are displayed as funnel plots summarising overall performance for each operator and cumulative funnel plots for an individual operator’s performance on a case series basis.Results The funnel plots for 5198 patients undergoing percutaneous coronary interventions showed an average observed rate for major adverse cardiovascular and cerebrovascular events of 1.96% overall. This was below the predicted risk of 2.06% by the logistic north west quality improvement programme risk score. Rates of in-hospital major adverse cardiovascular and cerebrovascular events for all operators were within the 3σ upper control limit of 2.75% and 2σ upper warning limit of 2.49%.Conclusion The overall in-hospital major adverse cardiovascular and cerebrovascular events rates were under the predicted event rate. In-hospital rates after percutaneous coronary intervention procedure can be monitored successfully using funnel and cumulative funnel plots with 3σ control limits to display and publish each operator’s outcomes. The upper warning limit (2σ control limit) could be used for internal monitoring. The main advantage of these charts is their transparency, as they show observed and predicted events separately. By this approach individual operators can monitor their own performance, using the predicted risk for their patients but in a way that is compatible with benchmarking to colleagues, encapsulated by the funnel plot. This methodology is applicable regardless of variations in individual operator case volume and case mix.     

An Integrated Case Study for Evaluating the Impacts of an Oil Refinery Effluent on Aquatic Biota in the Delaware River: Sediment Core Studies

The objectives of this study were to (1) determine if historical exceedences of oil and grease concentrations above the limits allowed in the National Pollution Elimination Discharge System permit for the wastewater treatment facility of Motiva Enterprises LLC Refinery, Delaware City, Delaware, could be determined from the sedimentary records of the Delaware River and, if so, (2) were the concentrations of contaminants high enough to be toxic to aquatic biota. Eighty-four surficial sediment samples, both within and outside the influence of the Refinery's discharge plume, were initially evaluated for their appropriateness for coring. Seven stations were ultimately selected for the historical core studies. Based on sediment type, radionuclide (Pb-210, Cs-137 and Be-7) geochronologies, and proximity of the cores to the Refinery, two cores were selected for more detailed polycyclic aromatic hydrocarbon (PAH) analyses. The rapid accumulation area of one core (located in the near-field of the discharge plume) had maximum total PAH (TPAH) concentration peaks at depths of 4 cm (1997; 3100 ng/g dw) and at 8.5–9 cm (1993; 3200 ng/g dw); the second core (located on the far-field periphery of the plume) had a maximum TPAH peak at 6.5 cm (1997; 3200 ng/g dw). In all cases, the maximum TPAH concentrations were below NOAA's Effects Range-Low concentration of 4022 ng/g dw for sediment biota toxicity. A chemical fingerprinting analysis of the PAHs in the two cores showed, however, that the PAHs present in the cores were predominantly pyrogenic PAHs consistent with Delaware River background PAHs. The core results are consistent with historic sediment PAH inputs in an industrial river system. PAH from the Refinery, if present, exist as a non-detectable increment to a high PAH baseline from many sources.     

Buffering an Acidic Stream in New Hampshire with a Silicate Mineral

Ground and pelletized Wollastonite (Wo; CaSiO₃) was added to a 50‐m reach of an anthropogenically acidified stream within the Hubbard Brook Experimental Forest, New Hampshire, to evaluate its buffering and restoration potential. The Wo was highly effective in raising the pH, acid‐neutralizing capacity (ANC), dissolved inorganic carbon (DIC), and Ca²⁺ concentrations of the stream water, but during the short duration of the experiment had no discernable effect on the stream biota. After initial, spike‐like fluctuations in pH and concentrations of ANC, DIC, and Ca²⁺, the relatively slow dissolution rates of the Wo dampened extreme concentrations and contributed to relatively long‐lasting (4 months) amelioration of streamwater acidity. Changes in concentrations of Ca²⁺, dissolved Si, ANC, and DIC were inversely related to streamflow. After several high, stream‐discharge events, concentrations quickly and consistently returned to pre‐event conditions.     

Characterization of an enriched lipoxygenase extract from Aspergillus niger in terms of specificity and nature of flavor precursors production

Aspergillus niger was grown for 6 days, and the harvested biomass was homogenized; the resultant supernatant, considered as the crude enzymatic extract, was enriched by ammonium sulfate precipitation. The extract was assayed for its lipoxygenase (LOX) activity using a wide range of polyunsaturated fatty acids (PUFAs), including linoleic, linolenic and arachidonic acids, as substrates. Two pH maxima were determined at 5.0, 10.5. The K_m and V_max values indicated that the microbial LOX displayed preferential substrate specificity towards linolenic acid at low pH. The microbial LOX demonstrated preferential substrate specificity towards free fatty acids over the acyl esters of linoleic acid. It was shown that the LOX activity of A. niger produced all monohydroperoxy regioisomers of the PUFAs, and there was a predominance of conjugated diene hydroperoxides. Significant production of the unconjugated 10-hydroperoxides of both linoleic and linolenic acids was obtained by the LOX activity. The amounts of 10-hydroperoxides ranged from 15 to 21% of total produced isomers, for linolenic and linoleic acids, respectively. The greatest proportion of the 10-regioisomer was attributed to the maximum activity at pH 5.0. Four major hydroperoxy-eicosatetraenoic acid (HPETE) regioisomers were isolated from the bioconversion of arachidonic acid, including the 8-, 9-, 12- and 15-HPETE, which accounted for approximately 97% of total isomers.     

A target-unrelated peptide in an M13 phage display library traced to an advantageous mutation in the gene II ribosome-binding site

Screening of the commercially available Ph.D.-7 phage-displayed heptapeptide library for peptides that bind immobilized Zn2+ resulted in the repeated selection of the peptide HAIYPRH, although binding assays indicated that HAIYPRH is not a zinc-binding peptide. HAIYPRH has also been selected in several other laboratories using completely different targets, and its ubiquity suggests that it is a target-unrelated peptide. We demonstrated that phage displaying HAIYPRH are enriched after serial amplification of the library without exposure to target. The amplification of phage displaying HAIYPRH was found to be dramatically faster than that of the library itself. DNA sequencing uncovered a mutation in the Shine-Dalgarno (SD) sequence for gIIp, a protein involved in phage replication, imparting to the SD sequence better complementarity to the 16S ribosomal RNA (rRNA). Introducing this mutation into phage lacking a displayed peptide resulted in accelerated propagation, whereas phage displaying HAIYPRH with a wild-type SD sequence were found to amplify normally. The SD mutation may alter gIIp expression and, consequently, the rate of propagation of phage. In the Ph.D.-7 library, the mutation is coincident with the displayed peptide HAIYPRH, accounting for the target-unrelated selection of this peptide in multiple reported panning experiments.     

Amine-reactive isobaric tagging reagents: requirements for absolute quantification of proteins and peptides

Amine-reactive isobaric tagging reagents such as iTRAQ (isobaric tags for relative and absolute quantitation) have recently become increasing popular for relative protein quantification, cell expression profiling, and biomarker discovery. This is due mainly to the possibility of simultaneously identifying and quantifying multiple samples. The principles of iTRAQ may also be applied to absolute protein quantification with the use of synthetic peptides as standards. The prerequisites that must be fulfilled to perform absolute quantification of proteins by iTRAQ have been investigated and are described here. Three samples of somatropin were quantified using iTRAQ and synthetic peptides as standards, corresponding to a portion of the protein sequence. The results were compared with those obtained by quantification of the same protein solutions using double exact matching isotope dilution mass spectrometry (IDMS). To obtain reliable results, the appropriate standard peptides needed to be selected carefully and enzymatic digestion needed to be optimized to ensure complete release of the peptides from the protein. The kinetics and efficiency of the iTRAQ derivatization reaction of the standard peptides and digested proteins with isobaric tagging reagents were studied using a mixture of seven synthetic peptides and their corresponding labeled peptides. The implications of incomplete derivatization are also presented.