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Innate Antimicrobial Activity of Nasal Secretions
Alexander M. Cole, Puneet Dewan, Tomas Ganz
Alexander M. Cole
Departments of Medicine and Pathology and the Will Rogers Institute for Pulmonary Research, UCLA School of Medicine, Los Angeles, California 90095
Puneet Dewan
Tomas Ganz
Minimally manipulated nasal secretions, an accessible form of airway surface fluid, were tested against indigenous and added bacteria by using CFU assays. Antimicrobial activity was found to vary between donors and with different target bacteria and was markedly diminished by dilution of the airway secretions. Donor-to-donor differences in electrophoresis patterns of nasal secretions in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (PAGE) and acid urea-PAGE analyses were readily observed, suggesting that polymorphic genes encode the secreted proteins. Three donors (of twenty-four total), whose nasal fluid yielded similar protein band patterns and did not kill indigenous bacteria, were determined to be heavy nasal carriers of Staphylococcus aureus. Their fluid was deficient in microbicidal activity toward a colonizing strain of S. aureus but the defect was corrected in vitro by a 1:1 addition of nasal fluid from noncarriers. The microbicidal activity of normal fluid was inactivated by heating it for 10 min to 100°C and could not be restored solely by the addition of two major nasal antimicrobial proteins, lysozyme and lactoferrin. Several other known antimicrobial proteins and peptides, including statherin, secretory phospholipase A2, and defensins, were identified in nasal secretions and likely contribute to their total antimicrobial properties. Nasal fluid may serve as a useful model for the analysis of lower-airway secretions and their role in host defense against airway colonization and pulmonary infections.
Host defense is a prominent function of nasal secretions. Among the components of the secretions, mediators of adaptive immunity include immunoglobulin A (IgA) and IgG, which are produced by plasma cells juxtaposed to submucosal glands (29). The immunoglobulins are thought to act in the lumen of the airway and on the mucosa to prevent the attachment and invasion of pathogenic organisms. Mediators of innate mucosal host defense are also found in nasal secretions and include substances that selectively disrupt bacterial cell walls and membranes, sequester microbial nutrients, or act as decoys for microbial attachment. Alexander Fleming discovered the intrinsic antimicrobial properties of human nasal secretions in 1922 and attributed them to lysozyme (8). Since then, additional components of nasal secretions have been identified, including lactoferrin, mucus glycoproteins, secretory leukoprotease inhibitor (SLPI), uric acid, peroxidase, aminopeptidase, immunoglobulins, and neutral endopeptidase (reviewed in references15 and 16). Systematic analysis of the individual and combined antimicrobial effects of these and other components has not been reported. Pathological alterations in antimicrobial properties of airway secretions may contribute to epithelial colonization by bacteria in such disorders as cystic fibrosis, chronic bronchitis, and chronic sinusitis.
The epithelial lining fluid covering the airways derives its proteins from plasma transudate, mucous and serous cells in submucosal glands, goblet cells, Clara cells, epithelial cells, and other cells within the mucosa (plasma cells, mast cells, phagocytes, and fibroblasts). The epithelial lining fluid is a bilaminar mucous layer consisting of an outer viscous gel (mucus) and a periciliary serous layer (42). Nasal epithelial cilia continually transport mucus with ensnared particulate matter and microbes toward the oropharynx, where they are swallowed. Consequently, in the normal individual, the nasal mucus blanket turns over every 10 to 20 min and must be replenished. However, fluid is relatively stagnant in the anterior portion of the nasal mucosa (15), and there the elimination of impacted microbes must be largely dependent on its intrinsic antimicrobial properties. This stagnant anterior microenvironment might serve as a useful model of torpid viscous secretions in the airways of patients with cystic fibrosis and other forms of bronchiectasis.
The limited available evidence suggests that nasal and bronchial secretions have many similarities in their composition (23). However, unlike bronchial secretions, nasal secretions can be easily collected and could provide a useful foundation for the study of other airway secretions. We reexamined the antimicrobial properties of human nasal secretions by using a CFU microassay, wherein bacteria are directly incubated with minimally manipulated nasal secretions. In contrast to recent studies that have focused on the antibacterial action of individual components of lower airway fluids (12, 27,35, 44), the analysis of minimally manipulated fluid affords the opportunity to assess the natural antimicrobial activity within the airways.
Collection of nasal secretions.Nasal secretions were collected from 24 healthy volunteer donors according to a protocol approved by the University of California at Los Angeles (UCLA) Institutional Review Board. From most donors, nasal secretions were collected by vacuum-aided suction, without chemical stimulation, to avoid introducing foreign substances into the nasal fluids. Gentle manipulation of a narrow rubber-tipped vacuum device inside the nasal passageways mildly stimulated nasal secretions. The range of secretion volumes collected varied between 150 μl and 14 ml, depending on the donor. For selected collections, the secretion of nasal fluids was induced by 25 to 50 mg of methacholine chloride (Spectrum Pharmaceuticals, Irvine, Calif.) administered into one nostril by an atomizing dosimeter. The range of secretion volume varied between 0.5 and 15 ml per donor. Secretions were stored at 4°C for prompt usage or at −20°C for longer-term storage. The microbicidal activity and protein profile by acid urea-polyacrylamide gel electrophoresis (AU-PAGE) of secretions stored for 2 weeks at 4°C or >1 month at −20°C was unchanged compared to fresh samples.
Minimally manipulated fluid.Vacuum-aspirated nasal fluid collected without methacholine was sonicated for 2 h at 4 to 8°C in a Branson 1200 sonicating water bath (Bransonic Cleaning Equipment Co., Shelton, Conn.) to disrupt the mucoprotein aggregates and to facilitate accurate handling. Unless otherwise stated, fluid samples with a high number (>105/ml) of indigenous bacteria were gamma-irradiated at 4 to 8 krads. Sonication and gamma-irradiation did not produce discernible changes in microbicidal activity (data not shown).
Cationic (poly)peptide extraction from acidified nasal fluid.Fluids collected by methacholine stimulation were solubilized by 1:20 dilution in 5% acetic acid for 2 h at 4°C. Fluid was cleared by centrifugation at 21,000 × g for 15 min, and the resulting supernatant stored at −20°C. The supernatant was diluted threefold with distilled water (dH2O) and stirred overnight with a weak cation-exchange carboxymethyl (CM) resin (Bio-Rad, Hercules, Calif.) to extract cationic peptides and proteins. The resin was washed once with 25 mM ammonium acetate (pH 6.8), poured into a 1-by-6-cm column, and eluted on a 0.1% acetic acid–0.1 M HCl gradient at 100 μl/min. Peak fractions were tested by radial diffusion assay with Escherichia coli ML-35p and Listeria monocytogenes (39). Active fractions were subjected to reversed-phase high-pressure liquid chromatography (HPLC) (60-min gradient of 5 to 60% acetonitrile in 0.1% trifluoroacetic acid [TFA]; 4.6-by-250-mm Vydac C18 column; The Separations Group, Hesperia, Calif.). Peak fractions were tested by a gel overlay assay with E. coli ML-35p and L. monocytogenes (39). Antimicrobial (poly)peptide bands from duplicate gels were transferred to Trans-Blot polyvinylidene difluoride (PVDF) membranes (Bio-Rad) for 1 h at 0.18 A, stained with amido black (Sigma), and destained with dH2O. Proteins of interest were excised and submitted for microsequencing.
Gel electrophoresis.Nasal secretions were separated on duplicate gels by AU-PAGE (40) and Tricine-sodium dodecyl sulfate (SDS)-PAGE (33). We used two-dimensional PAGE to improve the resolution of certain proteins and peptides for microsequencing. Nasal secretions were separated on an AU-PAGE gel as the first dimension and transferred to a reducing SDS-PAGE gel as the second dimension. Briefly, an entire lane of an AU-PAGE gel, consisting of electrophoresed nasal secretion proteins, was incubated at room temperature in a 1:1 mixture of 1× Laemmli buffer (25 mM Tris base; 0.19 M glycine; 0.1% SDS) and 3× denaturing sample buffer (0.17 M Tris, pH 8.8; 6% SDS; 21% glycerol; 0.15 M dithiothreitol; bromophenol blue to color) for 10 min. The gel slice was subsequently inserted in the preparative well of an SDS-PAGE gel, sealed with 1% agarose in cathode running buffer (0.1 M Tricine; 0.1 M Tris, pH 8.25; 0.1% SDS), and electrophoresed for 5 h at ∼110 V. Tricine-SDS-PAGE gels for protein sequencing were transferred to Trans-Blot PVDF membranes as described above, and proteins of interest were excised and submitted for microsequencing.
(Poly)peptide identification.Microsequencing was performed by N-terminal Edman degradation of AU-PAGE or reducing SDS-PAGE gels transferred to PVDF membranes (UCLA Peptide Sequencing Facility). In addition, purified (poly)peptides were analyzed in the UCLA Center for Molecular and Medical Mass Spectrometry by electrospray ionization mass spectrometry on the Sciex API III (Perkin-Elmer Corp., Foster City, Calif.) and matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry on the Voyager RP Instrument (PerSeptive Biosystems, Framingham, Mass.).
Western analysis.After AU-PAGE, gels were immunoblotted to Immobilon P-PVDF membranes (Millipore, Bedford, Mass.) for 20 min (for very cationic peptides) to 2 h (lactoferrin) in 0.7% acetic acid–10% methanol as described previously (40). The membranes were then incubated with either a 1:250 dilution of rabbit polyclonal anti-human lysozyme (Dako Corp., Carpinteria, Calif.), a 1:5,000 dilution of anti-human lactoferrin (Dako), a 1:1,000 dilution of anti-human beta defensin-1 (HBD-1) (40), a 1:1,000 dilution of anti-HBD-2 (24), or a 1:1,000 dilution of goat anti-human SLPI (R&D Systems, Minneapolis, Minn.) overnight at room temperature. After several washings in 1× TTBS (Tris-buffered saline plus 0.05% Tween 20), membranes were incubated with a 1:2,000 dilution of alkaline phosphatase-conjugated polyclonal goat anti-rabbit secondary antibody (lysozyme, lactoferrin, HBD-1, and HBD-2; Pierce, Rockford, Ill.) or rabbit anti-goat secondary antibody (SLPI; Organon Teknika Corp., West Chester, Pa.) for 1 h at room temperature. The membranes were then developed by using nitroblue tetrazolium–5-bromo-4-chloro-3-indolylphosphate (NBT-BCIP) as a chromogenic substrate. Serial dilutions of standard human milk lysozyme, human milk lactoferrin (Sigma), human recombinant SLPI (a gift from Pieter Hiemstra, University of Leiden), and HBD-1 and HBD-2 (24, 40) were used as positive controls and for semiquantitative studies.
Bacterial strains and culture conditions.ThePseudomonas aeruginosa strain, designated CF, is an isolate (37) from a cystic fibrosis patient; the isolate was generously donated by M. J. Welsh (University of Iowa). P. aeruginosa CF, E. coli ML-35p, L. monocytogenes EGD, Staphylococcus aureus (clinical isolate; UCLA Clinical Microbiology Facility), and wild-typeSalmonella typhimurium were cultured for 18 h at 37°C in 50 ml of 3% Trypticase soy broth (TSB; all strains are described in reference 21). Each strain was subcultured for 2.5 h immediately prior to use in antimicrobial assays in 50 ml of 3% TSB to obtain mid-logarithmic-growth phase. Subcultures were then centrifuged at 1,400 × g for 10 min and washed once in a salt buffer (referred to as 1× nasal buffer [NB]) that approximates the electrolyte composition of nasal secretions: Na, 85 mM; Cl, 97 mM; K, 20 mM; Ca, 1 mM; P, 10 mM; Mg, 0.5 mM; and S, 0.5 mM (19, 25, 37). The bacteria were collected at 1,400 × g for 10 min and resuspended in 1 ml of 1× NB. An optical density at 625 nm of 0.16 to 0.18 approximated 2.5 × 107 CFU/ml. A second CF mucoid strain H246, its nonmucoid revertant H247, a lipopolysaccharide (LPS) mutant strain H234, and their wild-type parent (PAO1) strain H103 were all generously donated by Robert E. W. Hancock (University of British Columbia, Vancouver, Canada). These strains (11) were cultured for 18 h at 30°C but otherwise were treated as described above.
CFU microassays.To study the effect of nasal fluid on indigenous nonfastidious aerobically growing nasal microbes, 20 μl of nasal secretions (not gamma-irradiated) was incubated in closed 1.5-ml Eppendorf tubes at 37°C for 0, 1, 3, and 24 h and then spread onto 3% TSB–1% agar (TSA) plates with or without 5% sheep blood (Microdiagnostics, Lombard, Ill.). Plates were incubated for 16 to 18 h at 37°C overnight to permit visible colony growth. To determine the effect of nasal secretions on exogenous bacteria, 300 to 600 bacterial CFU per 5 μl in 1× NB were incubated with 20 μl of nasal secretions or 1× NB. The entire sample, or dilutions thereof, was spread after 0, 1, 3, and 24 h of incubation at 37°C onto TSA plates and incubated overnight for 16 to 18 h. With the exception of S. aureus, the colonies formed by resident flora were readily distinguishable from the test organism and could be counted separately. The survival of exogenously added S. aureus was determined by the difference in counts from controls with only indigenous bacteria.
Screening of donors for S. aureus nasal carriage.Twenty-four donors were screened for nasal carriage ofS. aureus by using the Bacto-Staph Latex Test for agglutination (Difco, Detroit, Mich.). The terminal 2 cm of the left nares were swabbed with a sterile cotton swab and immediately streaked onto TSA plates with 5% sheep blood. After 18 h of incubation at 37°C, multiple colonies were individually tested for agglutination (positive for S. aureus) according to the manufacturer's instructions. Donors' nasal fluid samples, which exhibited numerous strongly positive colonies, were submitted for confirmatory microbiological testing (UCLA Clinical Microbiology Laboratory).
Lysozyme purification.To obtain large quantities of human lysozyme for reconstitution studies, 75 ml of human milk was acidified to 5% acetic acid, incubated overnight, and centrifuged 30 min at 10,000 × g. The cleared supernatant was then diluted 1:10 with dH2O, the pH was adjusted to 6.5 to 7.0 with ammonium hydroxide, and 1:100 (vol/vol) CM-Prep weak cation-exchange resin (Bio-Rad) was added and allowed to bind overnight at 4°C with gentle shaking. The resin was washed with 25 mM ammonium acetate (pH 6.8), extracted with 10% acetic acid, lyophilized to dryness, resuspended in 1% acetic acid, and fractionated on a P10 size exclusion column (Bio-Rad). The second of two peak fractions shown by absorbance at 220 nm was pooled, lyophilized to dryness, resuspended in 5% acetic acid, and subjected to reversed-phase C8 HPLC (Vydac). Lysozyme peak fractions, eluting at 40% acetonitrile–0.1% TFA, were lyophilized and resuspended in 0.01% acetic acid. Lysozyme purity and concentrations were confirmed by AU-PAGE, Western, and lysoplate analyses. A 75-ml milk sample yielded 4.5 mg of >90% pure lysozyme.
Lysoplate assay.Briefly, 0.5 mg of lyophilizedMicrococcus lysodeiktikus per ml was suspended in 66 mM sodium phosphate buffer (Na2HPO4-NaH2PO4, pH 7.0), combined with 1% agarose in 66 mM sodium phosphate, and poured in a level 9-by-9-cm square petri dish (30). Sixteen evenly spaced 3-mm wells were punched in the solidified agar, and 5 μl of sample was introduced into each well. The lysozyme enzymatic activity was determined by measuring the diameters of the zones of clearance relative to lysozyme standards.
Statistics.Bacterial colony counts (CFU assay) were performed in at least triplicate in each independent experiment, except when noted that the quantity of nasal secretions was limiting. Statistical analysis was performed on log10 CFU values to enhance the normal distribution. Sets of independent experiments were compared with a paired t test (SigmaStat; SPSS, Inc., Chicago, Ill.). Dilution experiments were analyzed by groupwise comparison with a two-factor repeated measures analysis of variance, followed by multiple pairwise comparisons by the Tukey Test (SigmaStat). Error bars represent the standard error of the mean and, unless otherwise stated, P values are listed as compared to counts at t = 0 min.
Microbicidal effect of nasal secretions on indigenous microbes.We first assayed the intrinsic antimicrobial properties of nasal secretions against indigenous microbes ex vivo. Minimally manipulated nasal secretions were incubated at 37°C, and microbial counts were measured by a CFU assay at 0, 1, 3 and 24 h (Fig.1). Although initially all samples contained 103 to 104 CFU/ml, in three samples (donors 4, 11, and 12), the CFU gradually decreased during incubation and none were detected by 24 h (Fig. 1A). In another sample (donor 9) CFU counts diminished with time but were still detectable at 24 h (Fig. 1A). In both cases, CFU counts were significantly decreased by 24 h compared to time zero (P < 0.03). In three other samples (donors 19, 20, and 24) microbes proliferated so that by 24 h a 1 to 2 log increase in CFU (P < 0.03) over input was observed. We next explored whether the dissimilar fates of resident bacteria in nasal secretions were due to differences in the strains of bacteria colonizing the nasal passageway and/or donor variations in antimicrobial or nutritive constituents.
The fate of indigenous bacteria in minimally manipulated nasal secretions. (A) The majority of nasal fluid samples were inhibitory to indigenous microbes. (B) Nasal fluid samples from a minority of donors were permissive for indigenous microbes. Asterisk indicates n = 1, due to insufficient amounts of nasal secretions from the indicated donors.
Antimicrobial activity of minimally manipulated nasal secretions against added bacteria.Panels of CFU microassays were employed to ascertain the effect of bacterial species and donor-to-donor variability on the antimicrobial capacity of nasal fluid. The differential effects of a single donor's secretions (donor 4) against multiple species and strains of bacteria is presented in Fig.2 and 3, while the various effects of multiple donors' secretions against an individual bacterial strain is shown in Fig.4. An inoculum of 2 × 104 to 4 × 104 CFU/ml was used to model the host-defense challenge by moderate numbers of inhaled microbes (Fig. 1A). To avoid selection bias, we included samples from donors from whom only scant amounts of nasal secretions could be collected, but their secretions were tested in a single assay (indicated by an asterisk).
Susceptibility of various strains of bacteria to a single donor's nasal fluid. Nasal secretions from donor 4 were subjected to a modified CFU microassay of nasal fluid targeting four strains of P. aeruginosa, E. coli ML-35p,S. typhimurium wild type, and S. aureusclinical isolate. (A) Secretions tested at three different times showed similar activities against P. aeruginosa CF between 0 and 3 h but varied in the amount of regrowth at 24 h. (B) Fluid incubated with four different strains of P. aeruginosashowed strain-specific antimicrobial activity. (C) Nasal secretions permitted the slow growth of E. coli and S. typhimurium; however, S. aureus was effectively cleared. Init, inoculum (input) at time zero for each sample tested, without the addition of nasal fluid.
Effect of fluid dilution on microbicidal activity. Six strains of bacteria were subjected to a CFU microassay of undiluted, 1:1-diluted, and 1:2-diluted minimally manipulated nasal fluid from donor 4. The CFU counts of S. aureus (clinical isolate) (A),E. coli (B), and S. typhimurium (C) were little affected. The antimicrobial effects of nasal secretions on S. aureus (isolate from nasal carrier donor 24) (D), P. aeruginosa CF (E), and P. aeruginosa H103 (F) were decreased with increasing dilution. The effects of undiluted, 1:1-diluted, and 1:2-diluted secretions are represented by closed circles, open triangles, and closed squares, respectively. The input inoculum (closed triangles) is shown for each graph.
Susceptibility of P. aeruginosa to multiple donors' nasal fluids. Activities of multiple donors' nasal secretions were tested individually against two strains of P. aeruginosa. Different donors' fluids were tested in a CFU microassay against a cystic fibrosis clinical isolate (A) and the wild-type strain H103 (B). Init, inoculum (input) at time zero for each donor tested, without the addition of nasal fluid. Asterisks indicate n = 1 , due to scant fluid from the indicated donors.
Nasal fluid samples obtained on different days from the same donor showed similar antimicrobial activity against P. aeruginosa CF at 3 h of incubation (Fig. 2A). By 24 h, minor regrowth of bacteria was observed in two of the three samples (b and c); however, the differences in CFU counts compared to input were not significant. Thus, variance in the composition of nasal secretions from the same donor may contribute to small fluctuations in bacterial regrowth.
The antimicrobial effect of nasal secretions from a single collection was seen to depend on the properties of the bacterial target (Fig. 2B). An LPS mutant strain, H234, and a mucoid CF strain, H246, were effectively cleared at 1 h with no regrowth at 24 h (P < 0.003). The PAO1 strain, H103, was not cleared but did not significantly grow throughout the observed time period. The nonmucoid revertant strain of H246, H247, slowly grew throughout the entire period of observation with a doubling time of approximately 8 h. Similar differences were observed in the effect of nasal fluid on non-Pseudomonas strains (Fig. 2C). S. aureus (clinical isolate) was effectively eliminated at 24 h compared with time zero (P < 0.003). In contrast,E. coli and S. typhimurium CFU counts slowly increased (P < 0.004 and P < 0.00007, respectively), with a doubling time of 10 to 16 h.
To assess the effect of dilution in a CFU microassay, donor 4's nasal secretions were diluted 1:1 and 1:2 with 1× NB. Dilution had little effect on antimicrobial activity against S. aureus (clinical isolate), E. coli, and S. typhimurium (Fig. 3A to C), as well as on P. aeruginosaH234, P. aeruginosa H246, and P. aeruginosa H247 (data not shown). However, S. aureus (nasal carrier isolate), P. aeruginosa CF, and P. aeruginosaH103 proliferated in diluted nasal fluid (Fig. 3D to F). In this latter group, the increased dilution resulted in significantly increased bacterial growth either at all of the timepoints tested (Fig. 3D, P < 0.02), at 1 and 3 h (Fig. 3E, P < 0.02), or at 3 h (Fig. 3F, P < 0.003). The observation that thinning of nasal fluids can increase bacterial proliferation indicates that the factors that determine microbial growth in nasal fluid are primarily suppressive and concentration limited.
To detect the variation of the antimicrobial effect between donors, minimally manipulated nasal secretions from seven different donors were tested in a CFU microassay against several strains of P. aeruginosa. Four strains were employed for this study: strains CF (CF isolate), H246 (CF isolate), H234 (LPS mutant), and H103 (wild type) (Fig. 4). Several patterns of antimicrobial effect emerged. All samples tested against strain CF decreased CFU counts by >2 log of the inoculum by 1 h (P < 0.0002) and nearly cleared the nasal secretions of exogenous bacteria by 3 h (Fig. 4A, P < 0.0004). Bacterial regrowth at 24 h appeared to be a donor-specific phenomenon. The wild type, H103, was at least transiently inhibited by nasal secretions from all donors tested, but regrowth at 24 h (P < 0.01) was again observed in secretions from several donors (Fig. 4B). The mucoid (alginate-producing) P. aeruginosa strain, H246, and the LPS-deficient mutant strain, H234, were intermediate in their susceptibilities to nasal secretions (data not shown). Thus, alginate production and LPS structure affect the sensitivity of bacteria to normal nasal fluid.
Identification of antimicrobial (poly)peptides in nasal secretions.We screened the nasal secretions by Western blotting with specific antibodies against known antimicrobial (poly)peptides (Table 1). Among the proteins so identified, lysozyme, lactoferrin, SLPI, and secretory phospholipase A2 (sPLA2) are antimicrobial components previously noted in nasal secretions. Prominent protein bands that could not have been identified by Western blotting were separated by AU-SDS/2-dimensional PAGE and transferred to PVDF membranes for protein sequencing. Several abundant (poly)peptides were identified as fragments of albumin and the light chain of IgG. Newly described cationic antimicrobial components of nasal secretions included two β-defensins (HBD-1 and HBD-2), an α-defensin (human neutrophil peptide 1 [HNP-1]), statherin, and β-microseminoprotein. The latter three peptides were identified from larger-scale (>5-ml) preparations of acid-extracted nasal secretions and were present at much lower concentrations than lysozyme, lactoferrin, or SLPI.
Eleven (poly)peptides identified in nasal secretions by Western analysis, microsequencing, and/or mass spectrometry
Variation in antimicrobial (poly)peptide content.Differences in the antimicrobial activities of nasal secretions could result from donor-to-donor variations in the protein composition of the secretions. Equal volumes of eight donors' nasal secretions were analyzed by AU-PAGE (Fig. 5A) and stained with Coomassie brilliant blue (Sigma) or analyzed by Tricine-SDS-PAGE and stained with silver stain (Fig. 5B). The variability of the protein patterns among the donors is readily seen. Although a unique protein fingerprint is seen in each donor's fluid, the electrophoretic patterns can be classified into three groups. Group "a" (donors 8, 20, and 24) had the highest protein concentration, group "b" (donors 9 and 11) had a moderate protein concentration, and group "c" (donors 2, 4, and 19) had the lowest overall protein concentration. Group a also had a number of low-molecular-weight (poly)peptides, several of which are possible antimicrobials. In agreement with previous reports (17), the total protein concentration in our nasal fluid samples (n = 3), as performed by the UCLA Clinical Laboratories by the pyrogallol red molybdate method (Synchron CX Microprotein Calibrator; Beckman Instruments, Inc., Brea, Calif.), was found to be 1 to 3 mg/ml. Concentrations of lysozyme, lactoferrin, and SLPI in crude nasal fluid (n = 5) as tested by semiquantitative Western analysis are given in Table 1. A β-defensin, HBD-2, was also detected by Western blot in three of five donors. Donor-to-donor differences in electrophoretic patterns of nasal secretions in SDS-PAGE and AU-PAGE may indicate that polymorphic genes encode many of the proteins of nasal secretions.
Donor-to-donor variability in the protein pattern of nasal secretions. (A) AU-PAGE of eight donor's nasal secretions shows variations in lactoferrin, albumin, β-microseminoprotein (beta-MSP), SLPI, lysozyme, and statherin concentrations. (B) Reducing Tricine-SDS-PAGE of eight donors' nasal secretions shows the variability in low-molecular-weight proteins. Superscripts indicate three donor groupings based on protein band similarities. The positions of identified components of nasal secretions are shown at the gel margins.
Nasal S. aureus carriage.Since the nasal fluid of donors 20 and 24 was permissive for the growth of indigenous bacteria (Fig. 1B) and the two protein band patterns were similar (Fig. 5), the indigenous bacteria were typed to identify microbiological similarities between these two donors. With a Bacto-Staph Latex rapid slide agglutination kit (Difco), both donors were found to have numerous colonies that tested positive for S. aureus. Twenty-two other donors' secretions were also subjected to the Bacto-Staph Latex agglutination test; only one of these donors had high numbers of colonies positive for S. aureus (donor 8), and two had a few colonies positive for S. aureus (donors 3 and 23). Samples of secretions from donors 8, 20, and 24, suspected of having high S. aureus colonization, were submitted to standard clinical microbiological typing, which confirmed this finding. Heavy S. aureus nasal carriage in 3 of 24 total donors (12.5%) is within the expected frequency based on previous estimates of carriage rates of 8 to 18% (18).
In principle, carriage could result either if the colonizing strains ofS. aureus are extraordinarily resistant to secretions or if the carriers' nasal fluid is favorable to S. aureusproliferation. S. aureus, isolated from a carrier's nose (donor 24), was tested in CFU microassays of carrier and noncarrier nasal secretions (Fig. 6A). The nasal fluids from four donors, two noncarrier (donors 4 and 11) and two carrier (donors 20 and 24, secretions gamma-irradiated), were tested. While the noncarriers' secretions were bacteriostatic (donor 4) or bactericidal (donor 11), both carriers' secretions permitted a 2- to 3-log increase in CFU counts of the S. aureus isolate compared to time zero (P < 0.05). This suggests that the S. aureus strain is not extraordinarily resistant to nasal secretions but that the carriers' secretions may be deficient in antimicrobial activity.
Nasal carriers of S. aureus are deficient in antimicrobial components that are restored by the addition of noncarrier secretions. (A) S. aureus, isolated from donor 24, was used as the target bacterium in a CFU microassay of nasal fluid from two noncarriers (donors 4 and 11) and two carriers (donors 20 and 24, gamma-irradiated fluid). While the noncarriers' fluids were bacteriostatic or bactericidal, the carriers' fluids did not inhibit bacterial growth. (B) The 1:1 mix of nasal secretions from an S. aureus carrier (donor 24) with fluid from a noncarrier (donor 4) is bactericidal to indigenous flora, while the mix of nasal secretions from an S. aureus carrier with heat-inactivated (boiling for 10 min) secretions from a noncarrier promoted the growth of indigenous bacteria.
Noncarrier and carrier nasal secretions, with their indigenous flora, were mixed to determine whether the antimicrobial activity of carrier fluid could be restored by the addition of noncarrier fluid. Figure 6B reveals that the indigenous flora of noncarrier secretions (donor 4) was killed throughout incubation (P < 0.01). When carrier fluid (donor 24) was incubated alone, its bacteria proliferated to 1.5 log units above the initial CFU count. Indigenous bacteria in carrier secretions incubated with heat-inactivated (boiling for 10 min) noncarrier fluid showed growth kinetics similar to that of carrier fluid incubated alone. However, a 1:1 (vol/vol) mixture of minimally manipulated carrier and noncarrier fluids was bactericidal for indigenous bacteria in the carrier's secretions (P < 0.02 compared to carrier fluid with or without the addition of heat-inactivated normal secretions). A similar pattern of activity was seen with the same noncarrier fluid (donor 4) and different carrier fluid (donor 20) (data not shown). A 1:1 (vol/vol) mixture of carrier (donor 20) and noncarrier fluids was bactericidal (P < 0.04 compared to either carrier fluid alone or carrier fluid mixed with heat-inactivated noncarrier fluid). Thus, the noncarrier nasal secretions contain antimicrobial substances that are either deficient or inactive in the carrier's nasal secretions.
Lysozyme and lactoferrin did not restore the microbicidal activity of heat-inactivated nasal secretions.Physiological concentrations of human milk lysozyme and human milk lactoferrin (Sigma), reported as the major antimicrobial agents in nasal secretions (32), were added to heat-inactivated fluid in an attempt to reconstitute the antimicrobial activity of nasal secretions (Fig.7). While unaltered nasal fluid was bactericidal, P. aeruginosa H246 was insensitive to heat-inactivated secretions. Lysozyme activity in heat-inactivated nasal fluid, as measured by the lysoplate assay, was found to be >25-fold less than in nonboiled control (n = 4; data not shown). The addition of 200 μg of lactoferrin per ml alone, 500 μg of lysozyme per ml alone, or the combination of 200 μg of lactoferrin and 500 μg of lysozyme per ml to heat-inactivated nasal fluid did not restore its antimicrobial activity (P < 0.03). The addition of lysozyme restored the enzymatic activity of nasal fluid towards Micrococcus lysodeikticus in the lysoplate assay (data not shown), but lysozyme added alone increased the growth of P. aeruginosa H246 at 24 h compared to growth in boiled nasal fluid (P < 0.04). The difference in the effect of lactoferrin alone and the lactoferrin-lysozyme combination was not statistically significant at 24 h compared to the boiled nasal fluid alone. Thus, other (poly)peptides besides lysozyme and lactoferrin contribute to the total microbicidal activity of nasal fluid.
Physiological concentrations of lysozyme and lactoferrin added to heat-inactivated nasal fluid (donor 4) did not restore microbicidal activity. While nonboiled fluid exhibited the expected bactericidal activity, heat-inactivated (boiling for 10 min) did not inhibit CFU growth. Physiological concentrations of lactoferrin and lysozyme, added separately and concurrently to heat-inactivated secretions, did not restore the antimicrobial activity.
Since the pioneering studies of Fleming (8), scant attention has been paid to the antimicrobial activity of nasal secretions and their role in the prevention of microbial colonization. We were motivated to reexamine the antimicrobial activity of nasal fluid because of recent evidence that the bacterial colonization of the airways in cystic fibrosis is due to a local defect in epithelial host defense (26). Our study confirms Fleming's observation that nasal secretions have intrinsic antimicrobial activity, but it also shows that this activity is precarious. Activity varies depending on the donor and the target bacteria; is often time limited, with regrowth observed by 24 h of incubation; and is ablated by dilution. Under normal conditions in vivo, the contributions of mechanical factors such as mucociliary clearance, phagocytic clearance, or sneezing may shorten the time available to microbes to adapt to the deleterious effects of nasal secretions. It is easy to conceive how the failure of one or more of these mechanisms could permit the colonization of airways by microbes.
Nasal S. aureus carriage has been identified as a risk factor for the pathogenesis of surgical and other hospital-associated infection. Persistent nasal carriers of S. aureus account for approximately 20% of the population, with 8 to 18% heavily colonized (>105 CFU/ml; reviewed in reference18). Although various epithelial and mucus host factors, such as surface glycoproteins and proteoglycans, have been shown to mediate binding, the precise adhesive molecules on host and bacteria have not been identified. S. aureus appears to attach to cell-associated and cell-free secretions (34) and to involve receptor sites of secretory IgA (3), glycolipids (20), and surfactant protein A (28). Various bacteria, including Staphylococcus epidermidis, are capable of reducing nasal ciliary activity in vitro (6). Enhanced adhesion and diminished mucociliary clearance could explain the retention of S. aureus within the nasal passageway but have no direct bearing on bacterial proliferation. Our observations suggest that the nasal fluid of at least some carriers is deficient in antimicrobial activity. Because of the natural donor-to-donor variation in the composition of nasal fluid, larger studies will be necessary to determine whether this mechanism is the sole or common determinant of nasal colonization by S. aureus and to identify the specific (poly)peptides or other substances that may be deficient in carriers.
Early colonization of cystic fibrosis airways predominantly involvesS. aureus, Haemophilus influenzae, orP. aeruginosa. The initially infecting P. aeruginosa organisms are typically nonmucoid; however, they become highly mucoid after continued exposure to the environment of the cystic fibrosis airways. Colonization with P. aeruginosa may induce further airway injury, perhaps due to destructive virulence factors, such as exotoxin A and proteases, and the recruitment of the host's neutrophils with their proteases, leading to the progressively destructive bronchitis and bronchiolitis. Some studies have attributedPseudomonas colonization in cystic fibrosis to abnormally glycosylated cell surface receptors with enhanced affinity for pseudomonal adhesive structures (13, 43). Other work suggests that abnormal mucin and the lack of hydration of respiratory secretions entraps bacteria, permitting colonization (22, 26,36). It has also been proposed that defects in intrinsic antimicrobial activity of airway secretions may be induced by alterations in the salt concentration due to defective chloride transport in cystic fibrosis (37). Our study of nasal secretions suggests that their antimicrobial activity is naturally variable from donor to donor and is often time limited. Factors, such as thick mucus or ciliary dysmotility, that delay the mechanical clearance of particulates from the airway may permit microbes to adapt to the damaging effects of secretions and to recover and resume growth. The ablation of the antimicrobial effects of nasal fluid by dilution ex vivo points to the possibility that reduced concentrations of antimicrobial substances due to abnormalities of fluid movement across epithelia could impair the antimicrobial activity of the airway secretions. The well-recognized phenotypic variability of cystic fibrosis among patients with identical mutations may in part be due to interactions with genes that control the (poly)peptide composition of nasal (and presumably other airway) fluid. Our observations on the variability of the protein patterns in nasal secretions are reminiscent of similar studies with human saliva, whose (poly)peptides are remarkably polymorphic (2). The effects of nasal (poly)peptide polymorphisms on host defense and their possible role as phenotypic modulators in cystic fibrosis should be a productive area for further investigation.
Nasal secretions contain antimicrobial proteins and peptides as a first-line host defense against microbial invaders. Lysozyme and lactoferrin, stored in and secreted from serous cells in nasal submucosal glands, are the most abundant antimicrobial proteins of nasal fluid. They are bactericidal alone for gram-positive bacteria and in combination for some gram-negative bacteria (5, 32). Given their strong combined antibacterial properties and since few other major microbicidal proteins have been previously described in nasal fluid, we expected they would reconstitute the antibacterial activity in heat-inactivated secretions. Our experiments suggest that other factors, or cofactors acting with lysozyme and lactoferrin, contribute to the antimicrobial properties of nasal fluid.
sPLA2 has been previously identified from methacholine-induced nasal lavage fluid and mucosa as a far less abundant (poly)peptide by both immunostain and enzymatic activity (1, 38). Ca2+-dependent antimicrobial activity of sPLA2 has been demonstrated against both gram-positive and gram-negative bacteria (reviewed in reference 10). In the present study we identified sPLA2 in unstimulated nasal fluid as a potential contributor to the total microbicidal activity of nasal fluid.
The current study is the first to identify various small cationic antimicrobial peptides in nasal secretions. HBD-1 has recently been characterized in urogenital tissues (40) and bronchoalveolar lavage (35), while HBD-2 is expressed at sites of inflammation (24, 35). HNP-1 (9) was likely a degranulation product of transudated neutrophils. Statherin, a small antimicrobial phosphoprotein previously identified in saliva (14), was also found to be present in nasal secretions. Another low-molecular-weight protein, β-microseminoprotein, has been localized to several human and rat mucosal tissues (7, 41) and was identified here as a component of nasal fluid (Table 1). Its detection in nasal fluid suggests that β-microseminoprotein is a ubiquitous antimicrobial protein secreted as a component of mucosal defense. We also acknowledge the possibility that anionic antimicrobial (poly)peptides contribute to the antimicrobial activity of nasal fluid, as observed in ovine bronchoalveolar lavage fluid (4); however, our fractionation methodology was not designed to detect such peptides. The abundance of antimicrobial peptides and proteins present in nasal secretions, coupled with the inability of lysozyme and lactoferrin to restore the microbicidal activity of heat-inactivated nasal fluid toward the test strain of P. aeruginosa, suggest that the antimicrobial activity of nasal fluid results from complex effects of its many components.
This work was supported by grants from the Cystic Fibrosis Foundation and the National Institutes of Health (HL46809 and AI40268).
We are grateful to Erika Valore and Christina Park for recombinant HBD-1 and -2 and polyclonal antisera and also for their guidance in protein analysis. We thank Jenny Lee for the purification of human milk lysozyme, Jishu Shi for his assistance in sequencing several peptides, and the UCLA Protein Microsequencing Facility, Mass Spectrometry Facility, and Clinical and Microbiological Laboratories for their invaluable help with this project. We also thank Edith M. Porter for continual discussions on microbiology and the properties of lysozyme.
Editor: J. R. McGhee
Received 9 February 1999.
Returned for modification 19 March 1999.
Accepted 2 April 1999.
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Infection and Immunity Jul 1999, 67 (7) 3267-3275; DOI:
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Home » News » Deep Forest Raceway Returns In Gran Turismo 7
Deep Forest Raceway Returns In Gran Turismo 7
Deep Forest Raceway has a long history in the Gran Turismo series. It made its debut all the way back in 1997, in the first entry of these now classic racing games. However, the racing track has been absent since 2013's Gran Turismo 6.
The PS4's Gran Turismo Sport was notable for missing out series mainstays such as Trial Mountain and Deep Forest Raceway, as it sought to include more FIA approved courses as it shifted to a more esports style. But the fictional track is making a comeback for Gran Turismo 7. Deep Forest Raceway will be making a return in the next Gran Turismo, and Polyphony Digital has even shown off a gameplay video of the race circuit.
With 15 corners and going through wooded areas, and crossing a ravine, Deep Forest Raceway was popular for its flowy lines and natural sights. Making its debut in the first Gran Turismo, it received a huge upgrade in graphics on the PS2, with light streaming through the trees wowing players at the time. But the two mile-plus race course was notably absent from Gran Turismo Sport despite being included in seven other Gran Turismo games.
Polyphony Digital has posted a two minute video of an onboard flying lap around Deep Forest Raceway, on the PS5, showing off the track as it will be available to drive in Gran Turismo 7. The video shows a Lamborghini Murciélago driving against 15 other competitors in the forthcoming game. The new GT also sees the return of the Alfa Romeo 8C Competizione, as spotted by Traxion, which was also last seen in Gran Turismo 6.
Those who've watched the gameplay video have been impressed with the upgraded lighting, realistic camera shakes, and tire sounds of the new title, in the comments. The sound made by the cars and the tires do sound like an upgrade from the PS4's Gran Turismo Sport and the reflections are also a clear improvement on the previous entry.
Gran Turismo 7 is due to launch on both the PS4 and PS5 on March 4, 2022.
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Brigham Young University professor Alex Jensen analyzed 282 families with teenage siblings for a study that appears in the Journal of Family Psychology. Favoritism in parenting is a complex topic for sure, but here are some important take-aways.
Yes, at least for some families. Jensen looked at perceived preferential treatment in different types of family dynamics. For families that aren't very close to each other – so-called "disengaged" families – favoritism was strongly associated with alcohol, cigarette and drug use by the less-favored children.
In these disengaged families, children who view themselves as slightly less favored were almost twice as likely to use alcohol, cigarettes or drugs. If the preferential treatment was perceived to be dramatic, the less favored child was 3.5 times more likely to use any of these substances.
In other words, favoritism appears to be the most problematic when love and support are generally scarce.
How can you tell who is the favorite?
When you ask people which sibling gets preferential treatment, their perception often doesn't match reality. But that's where things get tricky: Perceptions matter more.
For parents worrying about keeping score and managing perceptions of fairness, Jensen has some very simple advice.
That's based on what they saw in the data – the link between substance use and favoritism didn't exist among families that take a strong interest in each other.
Jensen also recommends that parents look for unique things that each of their children are trying to build into their identity.
This research was funded through a grant from the National Institute on Alcohol Abuse and Alcoholism awarded to Shawn D. Whiteman of Purdue.


What is a Condo Insurance Policy?
A condo insurance policy can vary widely from a homeowner's policy and we will help you understand the differences. In general, the association policy will cover the building and common areas, while you are responsible for the condominium's interior, your personal property, and potential liabilities. It is important to understand your responsibility as it relates to the association's insurance coverage as each association is different. The responsibilities for each party are provided in the CC&R's at the time of sale.


Chase Bank has kindly and diplomatically reached out to meet with me tomorrow evening about security measures at the Milwaukee Street branch. One main topic of our discussion will be the continued plans for an armed guard at the branch. This armed guard would be an off-duty Madison Police Department officer, for which Chase would cover the expense. I want to be sure that I have as much input from all of you as I can so I can best advocate for the neighborhood's interests and viewpoints.
One thing that current events have forced us to consider is how guns impact safety in all aspects of our lives. As we navigate how we respond to gun violence, there are a lot of pros and cons to having more armed guards to defend us, whether in our schools, or our neighborhood bank branches. This email would get impossibly long if I listed every single pro and con, but I'll try to get the main points out. I hope that we can unpack the bigger points of the discussion and that you can mull through everything and let me know where you come down on this.
A good number of our neighbors have reached out since the attempted robbery of about a month ago and shared that they feel greater sense of security knowing that there is a good guy/gal with a gun in a place where they bank. I take that seriously. Certainly feeling safe going about our neighborhood should be a basic expectation. If having an armed guard at Chase helps our neighbors feel safer, I definitely hear that. I understand the opinion of many that an armed guard is a deterrent to attempted robberies, and that a guard would almost never to need to actually fire their weapon.
I do wonder if an armed guard at this location truly makes us safer. I don't want to talk anyone out of their gut feeling on this because you know best what safety means for you. I am concerned that if there is one armed guard at the branch, that instead of one person trying to rob the bank without an actual weapon, as we saw frequently before now, that we might see a group of assailants, armed with powerful guns, attempt a robbery. We do an okay job setting up our officers with weapons, but we don't need to get into an arms race with would-be robbers. That would be terribly unsafe for everyone in the vicinity, not least our officers.
By the same token, I continue to be concerned with the possibility of gunfire in a residential area, regardless of who is firing the shots. There is a daycare just across the street, next to a senior care facility. Houses abut the bank property on the other 3 sides; some of our neighbors' living rooms are a couple of hundred feet from the bank lobby. MPD officers are highly trained on when to take a shot and would of course consider their surroundings as factors before firing their weapon. But there is no such thing as a perfect shot, and a bullet from a good guy's gun travels the same way as a bullet from a bad guy's gun. You know me, I'm not scared of much, but I would fear everyday that an attempted robbery and a stray bullet from either an assailant or even an officer would lead to tragedy in our district. While an armed guard, a highly trained MPD officer, would increase security in many ways, of course we must admit that more weapons equals more risk.
I'll bet she doesn't have that same opinion about armed security at Madison, Wisconsin city council meetings.


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Positive Infinity
Sailing the Last Voyage with Newton and Pascal
Posted on February 5, 2014 by Don Warrington
Granville Sewell on the Weaknesses of Darwinism
In view of yesterday's debate on the subject, this, which is the postscript for University of Texas at El Paso math professor Granville Sewell's book Analysis of a Finite Element Method: PDE/PROTRAN:
As I begin my 12th year of work on TWODEPEP (now PDE/PROTRAN ), I am intrigued by the analogy between the 11 year evolution of this computer code and the multi-billion year history of the genetic code of life, which contains a blueprint for a species encoded into billions of bits of information. Like the code of life, TWODEPEP began with primitive features, being capable of solving only a single linear elliptic equation in polygonal regions, with simple boundary conditions. It passed through many useful stages as it adapted to non-linear and time dependent problems, systems of PDEs, eigenvalue problems, and as it evolved cubic and quartic elements and isoparametric elements for curved boundaries. It grew a preprocessor and a graphical output package, and out-of-core frontal and conjugate gradient methods were added to solve the linear systems.
Each of these changes represented major evolutionary steps–new orders, classes or phyla, if you will. The conjugate gradient method, in turn, also passed through several less major variations as the basic method was modified to precondition the matrix, to handle nonsymmetric systems, and as stopping criteria were altered, etc. Some of these variations might be considered new families, some new genera, and some only special changes.
I see one flaw in the analogy, however. While I am told that the DNA code was designed by a natural process capable of recognizing improvements but incapable of planning beyond the next random mutation, I find it difficult to believe that TWODEPEP could have been designed by a programmer incapable of thinking ahead more than a few characters at a time.
But perhaps, it might be suggested, a programmer capable of making only random changes, but quite skilled at recognizing improvements could, given 4.5 billion years to work on it, evolve such a program. A few simple calculations would convince him that this programmer would have to rely on very tiny improvements. For example, if he could produce a billion random "mutations" per second (or, for a better analogy, suppose a billion programmers could produce one "mutation" per second each), he could not, statistically, hope to produce any predetermined 20 character improvement during this time period. Could such a programmer, with no programming or mathematical skills other than the ability to recognize and select out very small improvements through testing, design a sophisticated finite element program?
The Darwinist would presumably say, yes, but to anyone who has had minimal programming experience such an idea is preposterous. The major changes to TWODEPEP, such as the addition of a new linear equation solver or new element, required the addition or modification of hundreds of lines of code before the new feature was functional. None of the changes made during this period were of any use whatever until all were in place.
Even the smallest modifications to that new feature, once it was functional, required adding several lines, no one of which made any sense, or provided any "selective advantage", when added by itself.
Consider, by way of analogy, the airtight trap of the carnivorous bladderwort plant, which has a double sealed, valve-like door which is opened when a trigger hair is activated, causing the victim to be sucked into the vacuum of the trap (described by R.F.Daubenmire in "Plants and Environment," John Wiley and Sons, N.Y. 1947). It is difficult to see what selective advantage this trap provided until it was almost perfect.
This, then, is the fallacy of Darwin's explanation for the causes of evolution–the idea that major (complex) improvements can be broken down into many minor improvements. French biologist Jean Rostand, in "A Biologist's View" (William Heinemann Ltd., London, 1956) recognized this:
"It does not seem strictly impossible that mutations should have introduced into the animal kingdom the differences which exist between one species and the next…hence it is very tempting to lay also at their door the differences between classes, families and orders, and, in short, the whole of evolution. But it is obvious that such an extrapolation involves the gratuitous attribution to the mutations of the past of a magnitude and power of innovation much greater than is shown by those of today."
The famous "problem of novelties" is another formulation of the objection raised here. How can natural selection cause new organs to arise and guide their development through the initial stages during which they present no selective advantage, the argument goes. The Darwinist is forced to argue that there are no useless stages. He believes that new organs and new systems of organs arose gradually, through many small improvements. But this is like saying that TWODEPEP could have made the transition from a single PDE to systems of PDEs through many five or six character improvements, each of which made it work slightly better on systems.
It is interesting to note that this belief is not supported even by the fossil evidence. Harvard paleontologist George Gaylord Simpson, for example, in "The History of Life," Volume II of "Evolution after Darwin," (University of Chicago Press, 1960) points out:
"It is a feature of the known fossil record that most taxa appear abruptly. They are not, as a rule, led up to by a sequence of almost imperceptibly changing forerunners such as Darwin believed should be usual in evolution…This phenomenon becomes more universal and more intense as the hierarchy of categories is ascended. Gaps among known species are sporadic and often small. Gaps among known orders, classes and phyla are systematic and almost always large. These peculiarities of the record pose one of the most important theoretical problems in the whole history of life: Is the sudden appearance of higher categories a phenomenon of evolution or of the record only, due to sampling bias and other inadequacies?"
Another way of describing this same structure is expressed in a recent Life magazine article (Francis Hitching, "Was Darwin Wrong on Evolution?", April 1982, which concludes that "natural selection has been tested and found wanting") which focuses on the "curious consistency" of the fossil gaps:
"These are not negligible gaps. They are periods, in all the major evolutionary transitions, when immense physiological changes had to take place."
Unless we are willing to believe that useless, "developing" organs (and insect traps which could almost catch insects) abounded in the past, we should have expected the fossil structure outlined above, with large gaps between the higher categories, where new organs and new systems of organs appeared.
Nevertheless, despite the fact that the structure of the fossil record is the only argument against Darwin which has received much attention lately, this is not the real issue. The "problem of novelties" correctly states the real argument, but too weakly. Consider, for example, the human eye, with an aperture whose size varies automatically according to the light intensity, controlled by reflex signals from the brain; with a lens whose curvature varies automatically according to the distance to the object in view; and with a retina which receives the picture on color sensitive cells and transmits it, complete with coded intensity and frequency information, through the optic nerve to the brain. The brain superimposes the pictures from the two eyes and stores this 3D picture somehow in memory, and it will be able to search for and recall this image later and use it to recognize an older but familiar face in a different picture. Like TWODEPEP, the eye has passed through various useful stages in its development, but it contains a large number of features which could not reach usefulness in a single random mutation and which provided no selective advantage until useful (e.g. the nerves and arteries which service it), and many groups of features which are useless individually. The Darwinist may bridge the gaps between taxa with a long chain of tiny improvements in his imagination, but the analogy with software puts his ideas into perspective. The idea that all the magnificent species in the living world, or the human brain with its human consciousness, could have arisen from simple organic molecules guided by a natural process unable to plan beyond the next tiny mutation, is entirely comparable to the idea that a programmer incapable of thinking ahead more than a few characters at a time could, given a lot of time, design any sophisticated computer program.
I suggest that, with Jean Rostand, "we must have the courage to recognize that we know nothing of the mechanism" of evolution.
As someone who has put a few lines of code out these last two score, Sewell's observations on the subject make sense. This, written in 1985, anticipates at least two of the "hot button topics" this debate has engendered: the transitional fossils issue and the issue of irreducible complexity that Michael Behe made famous.
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New GOP Leaders of Arizona Legislature Lay Out Priorities
Dec 27, 2018 | Blog
Incoming House Speaker Rusty Bowers and incoming Senate President Karen Fann said raises for those workers is a top priority when the Legislature opens its 2019 session next month.
The two leaders diverge on some other top spending priorities for the surplus that is considered to be "one-time" funds.
Fann wants to put more money into the state rainy day fund to ensure there is money to pay for just-approved teacher raises if a recession hits.
Bowers is looking to use much of that cash to pay down a $930 million education "rollover." The budget trick pushes funding into the following fiscal year.
Paying down the rollover would immediately inject cash into schools that could be used to pay staff, repair buildings or buy new textbooks.
"That's just what we've owed them, $900 million-ish, for a long time," Bowers said. "I'd like to take care of a lot of that. If we could take care (of) all of it, I would, but I don't think we'll get that far."
The two leaders elected by Republicans in their respective chambers after the November election spoke to The Associated Press in separate interviews.
Their positions allow them to significantly influence what legislation moves through the House and Senate and negotiate budget items with Republican Gov. Doug Ducey.
Minority Democrats gained seats in the state House, which has a 31-29 split, but were unable to erode the Republicans' 17-13 Senate majority. The tighter House split gives Democrats more power, but if no GOP members break ranks, Republicans can pass a budget on their own.
Ducey will release his budget plan early next month.
Fann said the budget surplus, driven by improving revenue from an economy that has recovered from the Great Recession, can be a double-edged sword.
"The good is that we get to make some good decisions – the bad is when you have extra money everybody has great ideas how to spend that money," she said. "And it's important that we make sure that the bulk of it is one-time spending because we can't commit ourselves to something knowing full-well that we will be going through a recession again, just because we always do."
Despite both making raises for prison guards and Department of Public Safety troopers a priority, Fann and Bowers were less optimistic about giving raises to the remainder of the state's 32,000 employees.
The most recent state analysis, done in November 2017, shows state workers lagging private sector compensation by nearly 14 percent.
Bowers said he's focused on corrections and DPS, and "if there's enough for others that's great." Fann said she worries about committing to ongoing spending and wants to review state benefits packages as part of any discussion about raises.
A top priority for both is "conforming" the state income tax code to the federal tax code, which was overhauled to cut rates in late 2017. That will need to be done quickly, so taxpayers can file a simplified state return based on their adjusted federal gross income.
Simply adopting the federal code means Arizona would see a boost of state revenue, and GOP leaders see that as a tax increase, even though affected taxpayers will receive much bigger federal tax breaks.
Legislative budget analysts estimate the potential additional state revenue from conforming to the federal changes at $133 million to $236 million, while Ducey's administration estimates it between $180 million and $200 million.
"What I don't want is the need to have to file two forms." Bowers said. "And I would much prefer to have as much money as possible in the hands of the people."
Fann said the elimination of many deductions and the increase in the standard deduction will help many taxpayers but hurt some who heavily itemize, such as small businesses and Realtors. And no matter what lawmakers do, the complexity of taxes means it will be hard for lawmakers to quickly digest.
Ducey spokesman Patrick Ptak said the governor wants Arizona to conform to the federal changes and put the resulting additional state revenue from the 2018 tax year into the state's rainy day fund.
That will provide taxpayers with certainty about the 2018 tax year while letting the governor and lawmakers then work out what to do about future tax years, Ptak said.
Other top issues include adopting a plan addressing the state's Colorado River water shortage and a push to reform the criminal justice system.
Both lawmakers say they're upset with a new car registration fee adopted last year to free up money for highway repairs and schools. That was estimated to be $18 per vehicle, but when Ducey's administration announced its final fee last month, it was $32. That angered many Republicans who voted for the proposal, including Bowers and Fann, and the first bill filed for the upcoming session is a repeal.
"I will say that we are all very, very disappointed," Fann said. "We need to have a conversation about that, because that's not what was intended."
Ducey told reporters recently that the ongoing fee is part of last year's budget deal and he's not open to revisiting the issue.
"I hope he's not really nailed down on that," Bowers said.
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Law backed by Prescott Republican Senate President Karen Fann mandates 1st District designation remains with Prescott
Senator Karen Fann Recognized for Protecting Arizona's Workforce


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Wedding Bells.
Donna Courtney, daughter of Jimmy and Dolores or Ardcarney, Ruan, married Mortimer O'Sullivan, son of Margaret and Gerry of Inishannon, Co. Cork, in St. Mary's Church, Ruan, recently. Bridesmaids were Lisa Courtney, Martha Fitzpatrick and Rosaleen Finn. The groom's three brothers, Denis, Declan and Eoin were groomsmen. Fr. Pat O'Neill was celebrant. Following the ceremony, the couple and their families and friends enjoyed a reception at the Armada Hotel, Spanish Point.
Nathan on Stage.
Local boy, Nathan Bluett from Dysart, is playing Kurt - of the Von Trapp family, in Ennis Society's Musical production of "The Sound of Music", which is being staged until Sat. 10th March, in Glor. Nathan is a past pupil of Toonagh N.S. and is presently attending Gaelcholaiste an Chlair in Ennis, and has always had an interest in singing. He has previously appeared in "Oliver" and enjoys taking part in the productions.
40th Celebrations in Toonagh.
Forty years ago, Toonagh Hall and Sports Complex came into existence, with a great community effort, around the building which housed the old school in Toonagh. Over the years there have been many enjoyable evenings, with Socials, Marquees, set dancing classes, handball tournaments, youth clubs, meetings, yoga, flower arranging classes, keep fit classes, hurling and camogie training which have brought the community together. Some of the most memorable nights held in the Hall were the Friends of Toonagh Concerts, at which the cream of traditional music, song and dance appeared. This year, being a special anniversary, Thurs. 29th March has been earmarked for a return to the stage of many of those who have already performed in Toonagh, as well as new and young talent, with a Concert, starting at 8.00p.m., in a fundraiser for the upkeep of the hall and sports complex.
There has been a lot of work done there over the past two years, heating was upgraded, the hall itself was dry lined, a new kitchen installed, and these have brought the hall up to a good standard, however work needs to be done in the toilets, and that, and the general upkeep of the venue, with annual Insurance, electricity, etc. requires that there is money available.
It is hoped that the community will come out and support what is a very valuable asset to the people of the parish, young and old.


What if ive had a last-minute change of plans and cannot get to the theatre today?
If you find that you are unable to attend a performance at the last minute, we ask that you let us know and we will give your ticket to a student who may be waiting for rush tickets for that show. We cannot, however, issue a tax credit for tickets donated less than 24 hours before the scheduled performance.


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Intelinair and Indigo Named Ag Tech Start-Ups to Watch in 2018 by Agriculture.com
Champaign, IL (January 23, 2018) — Intelinair, an aerial imagery analytics company based in San Jose, CA and Champaign, IL, was named one of the 7 Ag Tech Start-Ups to Watch in 2018 this month by Agriculture.com.
Intelinair's AgMRI® platform, the leading aerial analytics solution for agriculture, alerts farmers to emergence issues, early-season weeds, nutrient deficiencies, weather damage, water stress, disease outbreaks and insect infestations, giving users the ability to make data-driven decisions that impact this season and beyond.
"We're excited to continue to provide valuable, actionable and profitable insights to growers into 2018," said Al Eisaian, Co-founder and CEO of Intelinair. "The strength of farmer know-how combined with frequent and powerful high-resolution imagery analytics is the future of a sustainable agriculture."
Among those named as ag tech start-ups to watch in 2018 is Indigo Ag, a company dedicated to harnessing nature to help farmers sustainably feed the plant. To date, Indigo Ag has raised over $300 million and is the top-funded ag tech start-up.
Since unveiling AgMRI® in 2017, Intelinair has quickly become the largest aerial monitoring and analytics company, rapidly growing from the 1.8 million acres monitored last year. Other significant advancements include updated user functionality through the John Deere Operations Center and superior resolution from 15 centimeters (6 inches) per pixel in 2017 to 10 centimeters (4 inches) per pixel for the 2018 season. The company is also expanding its service to the Brazilian market later this year.
Intelinair will be featured at the World Agri-Tech Innovation Summit March 20-21 in San Francisco, California where Co-founder and CEO, Al Eisaian, is scheduled to speak. More information can be found at www.worldagritechusa.com.
To learn more about the AgMRI® platform, contact Intelinair at 833-629-4674 or visit www.agmri.com.
About Intelinair
Intelinair, Inc., is an aerial imagery analytics company focused on agriculture that delivers actionable intelligence to help farmers make data-driven decisions to improve operational efficiency, yields, and ultimately their profitability. Intelinair combines the power of aerial imagery analytics through traditional computer vision and modern deep learning methodologies, agronomic science and user-friendly interface (mobile) technologies to deliver near real-time decision support to farmers. The company's flagship solution AgMRI® is a field health monitoring and early-warning system that enables farmers to manage their operations proactively and with confidence. The company was founded in 2015.
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Support for academic visits to the UK and Türkiye
Doughty-Wylie Scholarships
Two grants of up to £1,000 each are available to support postgraduate students in any field of the arts, humanities and social sciences to participate in fieldwork or other research activity in Türkiye. In addition to the grant, the BIAA will offer successful applicants free accommodation at the BIAA hostel in Ankara.
Applicants should be British citizens enrolled on a Master's or PhD programme at a recognised UK Higher Education Institution. Research may relate to any historical period up to and including contemporary Türkiye.
Check currently open calls for applications.
The Turkish and Black Sea Scholars' Fund
Support is available for scholars of postgraduate status and above from countries bordering the Black Sea to travel to the UK in order to visit a recognised UK Higher Education Institution and further their research interests (e.g. by studying a museum collection, using a university library and/or presenting papers at conferences/workshops).
Support is also available for scholars to travel to the Institute's premises in Ankara in order to further their research interests by using the collections held at the BIAA's centre for research excellence.
Applicants wishing to visit the UK are expected to have provisionally organised their research trip prior to applying for BIAA financial support, and are asked to confirm the participation of the host institution in the UK. Applicants wishing to visit the Institute in Ankara are encouraged to apply to visit during the winter months, when accommodation at the Institute's hostel in Ankara is more readily available. Awards up to a maximum of £500 are available.
Support is available to support scholars of postgraduate status and above from countries bordering the Black Sea. The funds must be taken up within one year of award. Applications will be considered by the BIAA Research Committee.
Call for applications usually announced in March. Check currently open calls for applications.
The Martin Harrison Fellowship
Offered in conjunction with Oxford University
With funds donated in memory of the late Professor Martin Harrison, the University of Oxford has instituted a programme of short-term Fellowships to enable Turkish scholars working in any area of the material and visual culture of Anatolia from the Prehistoric to the Ottoman period to come to the UK and visit Oxford University for a period of research. The Fellowship is to support a stay of up to 60 days, and has a maximum value of £1,200. Additonally, the British Institute at Ankara will provide a return airfare to London.
Fellowships are open to Turkish citizens resident in Türkiye holding a degree in Archaeology or related fields such as epigraphy, numismatics, ancient history and art history. Applicants should have completed two years of postgraduate research. Fellowships are intended for doctoral students as well as for assistant and associate professors. Competence in speaking and writing English is necessary.
Society for the Promotion of Hellenic Studies Fieldwork Award
Sponsored by the Society for the Promotion of Hellenic Studies.
A grant of up to £400 is available to enable an undergraduate or postgraduate student to participate in a fieldwork project that relates to Hellenic studies (in its widest sense).
Support is available to enable an undergraduate or postgraduate student to participate in a fieldwork project in Türkiye or the region of the Black Sea littoral. Applicants must be undergraduate or postgraduate students based in a UK Higher Education Institution.
David French Scholarships
In memory of archaeologist Dr David French, Director of the British Institute at Ankara from 1969-1985 and indefatigable explorer of Türkiye ancient and modern, his family have established two scholarships to be administered by the Institute.
Two travel grants of up to £1,000 each are available to support undergraduate or postgraduate students to visit Türkiye and extend their knowledge and understanding of the region's history, geography, culture and environment. In addition to the grant, the BIAA will offer successful applicants free accommodation at the BIAA hostel in Ankara.
Applicants should be enrolled on a Bachelor's, Master's or PhD programme at a recognised UK Higher Education Institution. The fieldwork or other research activity for which support is sought may relate to any historical period up to and including contemporary Turkey. Applications relating to an individual's first visit to Turkey for study purposes will be particularly welcomed.


Whether you've been with your partner for six months or 10 years, it's perfectly normal to go through a rough patch in your relationship. In fact, even the healthiest relationships have their ups and downs. After all, humans are complicated beings with dreams, feelings, desires and, of course, faults and imperfections.
Sometimes it's clear that a relationship isn't going to work out. Perhaps you spend more time arguing than you spend being nice to each other. Or perhaps you're simply incompatible and the romance isn't there. When this happens, it's often wise to go your separate ways and embark on a new journey apart.
But if you love each other and you're both committed to making things work, there could still be the potential for a bright future. By working together to improve the relationship you can often reach a point of happiness, trust, and tranquillity.
Sometimes you might need a little outside help to break free from toxic behaviour or negativity and this is where a psychic comes into the equation. By seeking the support of a gifted psychic, you can often identify problem areas you hadn't considered before and make necessary changes for a fresh start.
Communication plays a vital role in every relationship. Without strong communication skills, you may find yourself standing back and watching as your relationship crumbles around you.
Making an appointment to see a psychic is a great first step to admitting there is a problem. Your psychic will help you open up about your hopes and fears while encouraging you to share your concerns and even your annoyances with your partner. By addressing the things that are causing issues, you can work together to resolve them.
With the help of a psychic reading, you'll be able to take a closer look at the inner workings of your relationship while also gaining an insight into what could lie ahead. A reading won't tell you the future, but it will give you a sneak peek into what the journey ahead might hold – it's up to you and your partner to take control of your destiny and shape your life together.
Whether you or your partner have been unfaithful or one of you is battling irrational trust issues, a psychic reading could help you to rebuild your faith in one another.
Your psychic will help you understand how a lack of trust can affect your relationship and guide you through the reconciliation process. They'll also help you let go of harmful worries and emphasise the need to invite peace and confidence into your life.
By tapping into their powerful psychic intuition, your reader may be able to identify potential roadblocks that could cause issues for you and your partner in the future. From money troubles to conflicting ambitions, your psychic will let you know if they have any suspicions for your life ahead. If they do raise awareness of potential problems, there's certainly no need to call it a day. Instead, think of this as a wakeup call and an opportunity to work together to tackle any issues before they rear their ugly heads!


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Sympycnus difficilis är en tvåvingeart som beskrevs av Van Duzee 1930. Sympycnus difficilis ingår i släktet Sympycnus och familjen styltflugor. Inga underarter finns listade i Catalogue of Life.

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