Speciation in chestnut-shouldered fairy-wrens (Malurus spp.) and rapid phenotypic divergence in variegated fairy-wrens (Malurus lamberti): a multilocus approach.

The chestnut-shouldered fairy-wrens comprise a subgroup of four species in the genus Malurus (Passeriformes: Maluridae). Collectively, they are widespread across the Australian continent but phenotypic variation is strongly structured geographically in just one species, M. lamberti. Earlier phylogenetic analyses of this group have been limited to one or two individuals for each species and have not represented all currently recognised subspecies of M. lamberti. Historically, the taxonomy and nomenclature of the M. lamberti complex has been debated, in part because of morphological similarities among its subspecies and another member of the group, M. amabilis. We reconstructed the phylogeny of all four species of chestnut-shouldered fairy-wrens including all four subspecies of M. lamberti using a mitochondrial gene (ND2), five anonymous nuclear loci and three nuclear introns. Phylogenetic analysis of the mitochondrial ND2 gene nests M. amabilis within M. lamberti rendering the latter paraphyletic. Individual nuclear gene trees failed to reliably resolve each of the species boundaries or the phylogenetic relationships found in the mtDNA tree. When combined, however, a strongly supported overall topology was resolved supporting the monophyly of M. lamberti and its sister species relationship to M. amabilis. Current subspecific taxonomy of M. lamberti was not concordant with all evolutionary lineages of M. lamberti, nominotypical M. l. lamberti being the only subspecies recovered as a monophyletic group from mtDNA. Some genetic structuring is evident and potential barriers to gene flow are discussed.

A phylogenetic analysis using full-length viral genomes of South American dengue serotype 3 in consecutive Venezuelan outbreaks reveals a novel NS5 mutation.

Dengue virus currently causes 50-100 million infections annually. Comprehensive knowledge about the evolution of Dengue in response to selection pressure is currently unavailable, but would greatly enhance vaccine design efforts. In the current study, we sequenced 187 new dengue virus serotype 3 (DENV-3) genotype III whole genomes isolated from Asia and the Americas. We analyzed them together with previously-sequenced isolates to gain a more detailed understanding of the evolutionary adaptations existing in this prevalent American serotype. In order to analyze the phylogenetic dynamics of DENV-3 during outbreak periods; we incorporated datasets of 48 and 11 sequences spanning two major outbreaks in Venezuela during 2001 and 2007-2008, respectively. Our phylogenetic analysis of newly sequenced viruses shows that subsets of genomes cluster primarily by geographic location, and secondarily by time of virus isolation. DENV-3 genotype III sequences from Asia are significantly divergent from those from the Americas due to their geographical separation and subsequent speciation. We measured amino acid variation for the E protein by calculating the Shannon entropy at each position between Asian and American genomes. We found a cluster of seven amino acid substitutions having high variability within E protein domain III, which has previously been implicated in serotype-specific neutralization escape mutants. No novel mutations were found in the E protein of sequences isolated during either Venezuelan outbreak. Shannon entropy analysis of the NS5 polymerase mature protein revealed that a G374E mutation, in a region that contributes to interferon resistance in other flaviviruses by interfering with JAK-STAT signaling was present in both the Asian and American sequences from the 2007-2008 Venezuelan outbreak, but was absent in the sequences from the 2001 Venezuelan outbreak. In addition to E, several NS5 amino acid changes were unique to the 2007-2008 epidemic in Venezuela and may give additional insight into the adaptive response of DENV-3 at the population level.

Genetic analysis of threatened Australian grayling Prototroctes maraena suggests recruitment to coastal rivers from an unstructured marine larval source population.

Population genetic variation of Australian grayling Prototroctes maraena was examined to determine whether the dispersal strategy of this amphidromous species favours retention of larvae and juveniles in close proximity to their natal river, or mixing of populations via marine dispersal. Variation in microsatellite and mitochondrial DNA markers was unstructured and differentiation was indistinguishable from zero across four coastal rivers spanning approximately one-quarter of the continental range of the species. This result indicates that the marine larval and juvenile phase probably facilitates extensive gene flow among coastal rivers and agrees with a previous analysis of otolith chemistry that suggested larvae probably move into the sea rather than remain in estuaries. It appears likely that the dispersal strategy of P. maraena would enable recolonization of rivers that experience localized extinction provided that connectivity between freshwater habitats and the sea is sufficient to permit migration and that enough source populations remain intact to support viability of the wider population.

Alternative indicator bacteria analyses for evaluating the sanitary condition of beef carcasses.

Sponge samples were obtained from 47 (study 1) and 32 (study 2) beef carcasses in a small plant over 6 months. In study 2, slaughter equipment surfaces were also sampled. In study 1, the Petrifilm method was used to count presumptive Escherichia coli and spread plating on kanamycin esculin azide (KEA) agar with and without 40% added bile was used to count presumptive Enterococcus spp. Qualitative testing for presumptive E. coli and Enterococcus spp. in study 1 was done using lauryl sulfate tryptone broth (LST) + 4-methylumbelliferyl-beta-D-glucuronide (MUG) and KEA + 40% bile broth, respectively. In study 2, LST + MUG was used as a most probable number (MPN) method along with the Petrifilm method. In the two studies, 8 (17.0%) and 11 (34.4%) carcasses were contaminated with presumptive E. coli; all but one contaminated carcass contained <1 CFU/cm2. Presumptive Enterococcus spp. were recovered from 15 carcasses (31.9%) in study 1, but the KEA + 40% bile agar method lacked specificity (only 31.3% of isolates confirmed as Enterococcus spp.) The LST + MUG and Petrifilm methods were significantly (P < 0.05) related in terms of detecting presumptive E. coli, but the presence of presumptive Enterococcus spp. was not significantly related to the presence of presumptive E. coli. However, on slaughter plant equipment in Study 2 there was a statistically significant (P < 0.05) relationship between the presence of presumptive E. coli and presumptive Enterococcus spp. In study 2, there was no significant (P < 0.05) difference in numbers of presumptive E. coli (obtained using Petrifilm) on carcasses chilled 1 day (n = 16) and 7 days (n = 16), although more of the 7-day carcasses were contaminated (five and seven carcasses, respectively). For samples testing positive for presumptive E. coli, the 95% confidence intervals obtained using the LST + MUG MPN method included the Petrifilm value for all but one sample.

Distribution of active protein kinase C in smooth muscle.

To localize activated protein kinase C (PKC) in smooth muscle cells, an antibody directed to the catalytic site of the enzyme was used to assess PKC distribution by immunofluorescence techniques in gastric smooth muscle cells isolated from Bufo marinus. An antibody to vinculin was used to delineate the cell membrane. High-resolution three-dimensional images of immunofluorescence were obtained from a series of images collected through focus with a digital imaging microscope. Cells were untreated or treated with agents that increase PKC activity (10 microM carbachol for 1 min, 1 microM phorbol 12-myristate 13-acetate (PMA) for 10 min), or have no effect on PKC activity (1 micrometer 4-alpha phorbol, 12,13-didecanoate (4-alpha PMA)). In unstimulated cells, activated PKC and vinculin were located and organized at the cell surface. Cell cytosol labeling for activated PKC was sparse and diffuse and was absent for vinculin. After treatment with carbachol, which stimulates contraction and PKC activity, in addition to the membrane localization, the activated PKC exhibited a pronounced cytosolic fibrillar distribution and an increased total fluorescence intensity relative to vinculin. The distributions of activated PKC observed after PMA but not 4-alpha PMA were similar to those observed with carbachol. Our results indicate that in resting cells there is a pool of activated PKC near the cell membrane, and that after stimulation activated PKC is no longer membrane-confined, but is present throughout the cytosol. Active PKC appears to associate with contractile filaments, supporting a possible role in modulation of contraction.

Length changes of the hamstrings and adductors resulting from derotational osteotomies of the femur.

Derotational osteotomies of the femur are frequently performed to treat persons with cerebral palsy who walk with excessive internal rotation of the hip. However, whether these procedures stretch or slacken the surrounding muscles appreciably is unknown. Determination of how muscle lengths are altered by derotational osteotomies is difficult because the length changes depend not only on the osteotomy site and the degree of derotation, but also on the anteversion angle of the femur and the rotational position of the hip. We have developed a three-dimensional computer simulation of derotational osteotomies, tested by anatomical experiments, to examine how femoral anteversion, hip internal rotation, and derotation affect the lengths of the semitendinosus, semimembranosus, biceps femoris long head, adductor longus, adductor brevis, and gracilis muscles. Simulation of derotational osteotomies at the intertrochanteric, subtrochanteric, or supracondylar levels decreased the origin-to-insertion lengths of the hamstrings and gracilis in our model by less than 8 mm (1.8%). Hence, the lengths of the hamstrings and gracilis are not likely to be altered substantially by these procedures. The origin-to-insertion lengths of the adductor longus and adductor brevis decreased less than 4 mm (1.9%) with subtrochanteric correction in our model, but the length of adductor brevis increased 8 mm (6.3%) with 60 degrees of intertrochanteric derotation. These muscles are also unlikely to be affected by derotational osteotomies, unless a large degree of intertrochanteric derotation is performed.

Common carotid artery flow velocity measurements in the newborn period with pulsed Doppler technique.

Using a commercial pulsed Doppler (PD) system we measured common carotid artery (CCA) flow velocities in 19 healthy and 6 stable preterm infants and computed volume flow rates based on the time-averaged flow velocities and vessel diameter. The mean (+/- SEM) time-averaged CCA velocities in the term and preterm infants were 33.04 +/- 3.0 and 23.3 +/- 1.3 cm.s-1, respectively. The total flow volume was significantly higher in the term as opposed to preterm infants: 126 +/- 11.2 vs. 55.6 +/- 7.7 ml/min (p less than 0.01). The body-weight-normalized flow volume, however, was not statistically significantly different between the two groups of infants. Several technical and practical limitations still exist to compute cerebral blood flow (CBF) volume accurately. In a puppy model we measured the CCA flow volume by electromagnetic flowmeter technique, and the PD device simultaneously. The correlation coefficient between 32 pairs of measurements was 0.93 (p less than 0.005). We conclude that commercial PD devices provide accurate velocity data, and under experimental conditions, the computed volume flow rates are accurate. Although in clinical situations reasonably accurate CBF values can be estimated, refinements in technology are needed to adopt this method as a possible means of measuring CBF at the bedside.

The pathogenesis of experimental pulmonary histoplasmosis. Correlative studies of histopathology, bronchoalveolar lavage, and respiratory function.

A murine model of acute pulmonary histoplasmosis was employed to study the pathogenesis of the disease process by means of histopathology, bronchoalveolar lavage, and respiratory function tests. These studies were performed on C57BL/6 mice from 8 h to 8 wk after intranasal inoculation of 10(5) yeast forms of Histoplasma capsulatum and on age-matched control animals that received saline only. At Week 1, the histopathology was characterized by subacute inflammation consisting of polymorphonuclear leukocytes (PMN), lymphocytes, and macrophages that infiltrated the interstitium around small bronchioles and adjacent alveoli. At Weeks 2 and 4, the infiltrates were comprised predominantly of lymphocytes and macrophages; noncaseating granulomas were present at Week 2. Aggregates of lymphoid cells were prominent along the bronchial tree and in perivascular distribution. Those in close contact with bronchiolar epithelium resembled hyperplastic bronchus associated lymphoid tissue. Quantitative studies of cells in the BAL fluid revealed a large influx of PMN at Week 1 with return to normal range by Week 2. At this time there was a significant (p less than 0.02) increase in lymphocytes that persisted through Week 8, although histopathologic changes were minimal in lung at this time. A significant decrease in the DLCO/TLC at Week 2 in association with a normal vital capacity indicated impairment of respiratory function secondary to the alveolitis induced by H. capsulatum infection rather than a reduction of lung volume. This model offers promise for additional correlative studies of lymphocyte subsets in lung tissue and alveolar spaces as well as of the functions subserved by these respective populations.

Toluene diisocyanate-induced airway hyperreactivity and pathology in the guinea pig.

We assessed the nature and progression of airway mucosal disease and histaminic reactivity in English short-haired guinea pigs at 2, 24, 72, 168, and 504 hours after toluene diisocyanate (TDI) exposure (4 hours of 3 ppm of TDI for 5 consecutive days). To also determine whether TDI-specific, IgE-like antibodies developed in TDI-exposed animals, passive cutaneous anaphylaxis testing was done 28 days after TDI. Bronchial reactivity was determined serially by measuring specific airway conductance as a function of increasing doses of aerosolized histamine in six exposed and three control animals studied intact and unanesthetized. The remaining 10 exposed and 10 control guinea pigs were sacrificed in groups of two at each time point to obtain airway tissue for light microscopic examination. We found that airway hyperreactivity to histamine occurred after TDI in all animals tested. It was maximal 2 hours after the 5-day exposure and remitted by 72 hours. In addition, marked airway obstruction occurred after TDI that persisted for at least 168 hours. There were dramatic signs of airway mucosal damage associated with the bronchial hyperreactivity that included substantial decreases in epithelial cilia, mucin content, and mast cells, as well as squamous metaplasia, numerous mitotic figures, and a prominent polymorphonuclear leukocytic infiltrate. Passive cutaneous anaphylaxis tests in exposed animals were negative. Our results suggest that TDI-induced bronchial hyperreactivity may be related to airway mucosal injury and inflammation.

Determination of sex with a discriminant analysis of new pelvic bone measurements: Part II.

The pelves of 100 white skeletons were measured on both sides for the following: (1) length from the superiormost aspect of the pubic symphysis to the nearest rim of the acetabulum (PS-A), (2) length from the highest point of the pubic tubercle to the nearest rim of the acetabulum (PT-A), (3) acetabular diameter (AD), (4) the vertical distance from the anterior aspect of the ischial tuberosity to the farthest rim of the acetabulum (IT-A), and (5) greatest femur head diameter. From these, three indices were derived: AD/PS-A (acetabulum/pubis index), AD/PT-A (acetabular diameter/pubic tubercle-acetabular rim index), and IT-A/PS-A (ischium-acetabulum height/pubic symphysis-acetabular rim index). The left AD/PS-A ratio and left IT-A height proved statistically to be of greatest discriminating value. Using these two variables, a discriminant function was derived which correctly separated 98% of our sample. The acetabulum/pubis ratio alone correctly assigned 95%. With either the discriminant function analysis of two variables or the acetabulum/pubis index as a single predictor, 97% of our sample of known sex was correctly identified if all specimens that fell within a doubtful or overlapping range of values were sorted by femur head diameter.

Determination of sex with a discriminant analysis of new pelvic bone measurements: Part I.

The pelves of 100 black skeletons were measured on both sides for the following: (1) length from the superiormost aspect of the pubic symphysis to the nearest rim of the acetabulum (PS-A), (2) length from the highest point of the pubic tubercle to the nearest rim of the acetabulum (PT-A), (3) acetabular diameter (AD), (4) the vertical distance from the anterior aspect of the ischial tuberosity to the farthest rim of the acetabulum (IT-A), and (5) greatest femur head diameter. From these, three indices were derived: AD/PS-A (acetabulum/pubis index), AD/PT-A (acetabular diameter/pubic tubercle-acetabular rim index), and IT-A/PS-A (ischium-acetabulum height/pubic symphysis-acetabular rim index). The left AD/PS-A ratio and left IT-A height proved statistically to be of greatest discriminating value. Using these two variables, a discriminant function was derived which, followed by sorting with femur head diameter, accurately classified 97% of our sample. The acetabulum/pubis index alone with subsequent sorting by femur head diameter correctly assigned 96% of our sample. While this does not represent an improvement of predicatability over similar methods using the ischium/pubis index, measurements required for the acetabulum/pubis index are more easily defined and should, therefore, reduce the chance of observer error.