Sampling large geographic areas for rare species using environmental DNA: a study of bull trout Salvelinus confluentus occupancy in western Montana.

This study tested the efficacy of environmental DNA (eDNA) sampling to delineate the distribution of bull trout Salvelinus confluentus in headwater streams in western Montana, U.S.A. Surveys proved fast, reliable and sensitive: 124 samples were collected across five basins by a single crew in c. 8 days. Results were largely consistent with past electrofishing, but, in a basin where S. confluentus were known to be scarce, eDNA samples indicated that S. confluentus were more broadly distributed than previously thought.

Marker placement for movement analysis in scoliotic patients: a critical analysis of existing systems.

Optoelectronic movement analysis systems has provided an opportunity for a detailed study of both normal and abnormal human walking and has contributed to the planning and documentation of corrective surgical procedures. The majority of reported studies have been on the study of lower limbs which, consequently, have received most attention in movement analysis. In contrast, movement of the trunk and pelvis, important in the identification of spinal mobility and maintaining posture, have received limited attention in relation to clinical conditions such as scoliosis. Any movement analysis requires the identification of anatomical landmarks which are essential contributing factors to the accuracy of the analysis. While there are a plethora of studies on marker placements for the lower limbs, there is a paucity of information on the marker locations for spinal analysis. Present study examines a set of markers previously reported in the literature and examines their usefulness in scoliotic gait analysis. The findings highlight the drawbacks in previously reported techniques and leads to the proposal of a new marker set for spine and back movement analysis.

Potential exposure to Australian bat lyssavirus is unlikely to prevent future bat handling among adults in South East Queensland.

Despite ongoing public health messages about the risks associated with bat contact, the number of potential exposures to Australian bat lyssavirus (ABLV) due to intentional handling by members of the general public in Queensland has remained high. We sought to better understand the reasons for intentional handling among these members of the public who reported their potential exposure to inform future public health messages. We interviewed adults who resided in a defined geographic area in South East Queensland and notified potential exposure to ABLV due to intentional handling of bats by telephone between 1 January 2012 and 31 December 2013. The participation rate was 54%. Adults who reported they had intentionally handled bats in South East Queensland indicated high levels of knowledge and perception of a moderately high risk associated with bats with overall low intentions to handle bats in the future. However, substantial proportions of people would attempt to handle bats again in some circumstances, particularly to protect their children or pets. Fifty-two percent indicated that they would handle a bat if a child was about to pick up or touch a live bat, and 49% would intervene if a pet was interacting with a bat. Future public health communications should recognize the situations in which even people with highrisk perceptions of bats will attempt to handle them. Public health messages currently focus on avoidance of bats in all circumstances and recommend calling in a trained vaccinated handler, but messaging directed at adults for circumstances where children or pets may be potentially exposed should provide safe immediate management options.

Comparison of stored and secreted rat pancreatic digestive enzymes by mass spectrometry: alpha-amylase.

As part of a continuing effort to better understand the mechanisms of protein secretion, we compared the mass of pancreatic digestive enzymes, in resting and stimulated states, both in secretion and in the zymogen granule to determine whether their secretion is accompanied by chemical modification. Mass spectra were obtained applying the electrospray method on samples separated by reverse-phase HPLC. We report here our results for alpha-amylase (1,4-alpha-D-glucan glucanohydrolase EC 3.2.1.1). The data illustrate structural differences between states and compartments for this enzyme. Multiple isozymes were identified from the mass spectra, varying roughly from 52 to 60 kDa. On the basis of mass comparisons, not all of the products seen in the zymogen granule were found in secretion, nor were all secreted isoforms in the granule. Stimulation of protein secretion with a cholinergic agonist, led to time-dependent changes in the number and masses of isoforms in secretion, leaving only one of five resolvable forms in the granule. Only one form, 55.5 kDa, was found in all samples, granule and secretion. In addition to these differences, microheterogeneities of 400 Da or less were observed. The data suggest the differential or non-parallel release of different amylase forms and their chemical modification during the secretion process. As such, release appears to involve a third, intermediate compartment, between zymogen granule to ductal space, such as the cytoplasm, in which chemical modification takes place.

Evidence of micro-continent entrainment during crustal accretion.

Simple models involving the gradual outboard accretion of material along curvilinear subduction zones are often inconsistent with field-based evidence. A recent study using 3-D geodynamic modelling has shown that the entrainment of an exotic continental fragment within a simple subduction system can result in a complex phase of growth. Although kinematic models based on structural mapping and high-resolution gravity and magnetic maps indicate that the pre-Carboniferous Tasmanides in southeastern Australia may have been subjected to this process, to date there has been little corroboration from crustal scale geophysical imaging. Here, we apply Bayesian transdimensional tomography to ambient noise data recorded by the WOMBAT transportable seismic array to constrain a detailed (20 km resolution in some areas) 3-D shear velocity model of the crust beneath southeast Australia. We find that many of the velocity variations that emerge from our inversion support the recently developed geodynamic and kinematic models. In particular, the full thickness of the exotic continental block, responsible for orocline formation and the tectonic escape of the back arc region, is imaged here for the first time. Our seismic results provide the first direct evidence that exotic continental fragments may profoundly affect the development of an accretionary orogen.

Aging: effects on the prostaglandin production by skeletal muscle of male rhesus monkeys (Macaca mulatta).

Prostaglandin (PG) production from [1-14C] arachidonic acid was measured in homogenates of sartorius muscle of aged (more than 20 years) and young adult (7 to 11 years) rhesus monkeys. Total production by the aged series (3.21 +/- 0.31 (S.E.) nmol PG per g N per min) was about two times that of the young series (1.76 +/- 0.14 (S.E.) nmol per g N per min). Epinephrine-stimulated PG production was also twice as great in the aged muscle series as in the young adult series (7.22 +/- 0.74 (S.E.) and 3.54 +/- 0.52 (S.E.) nmol per g N per min). This difference was mainly due to greater production of 6-keto-PGF1 alpha and PGF2 alpha. In both series, the addition of 500 microM epinephrine to the assay media significantly increased production of all types of PGs measured (6-keto-PGF1 alpha, PGF2 alpha, PGE2 plus thromboxane B2 [TXB2], the stable breakdown product of TXA2, and PGD2). The absolute increases in all types of PGs studied in the presence of epinephrine were significantly greater in the aged muscle, with the exception of PGF2 alpha. The distribution patterns of the various types of PGs studied were similar in all series, except for 6-keto-PGF1 alpha; this PG accounted for a greater percentage of the total production by aged muscle than by young adult muscle (p less than 0.05).

Method for screening peptide fragment ion mass spectra prior to database searching.

A methodology is described for screening fragment ion spectra of peptides prior to database searching for protein identification. A software routine written in the Perl programming language was used to analyze data from previous Sequest database searches and develop a set of statistical descriptors that could be used to identify spectra not likely to yield useful results in a database search. A second Perl program used an evolutionary algorithm to optimize the criteria for each statistical descriptor and generate a formula for determining spectral quality. This formula was used by a third Perl program to screen data sets from four independent liquid chromatography tandem mass spectrometry runs. On the average, use of the screening program reduced the time required for a database search by 1/2 with little loss of useful information from the database search results.

Importance of matrix:analyte ratio for buffer tolerance using 2,5-dihydroxybenzoic acid as a matrix in matrix-assisted laser desorption/ionization-Fourier transform mass spectrometry and matrix-assisted laser desorption/ionization-time of flight.

Many biological samples destined for matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) contain buffers. The presence of these buffers often inhibits the ability to obtain spectra. Here, the results of a study of the effects of six different buffers on spectra of three representative small proteins are reported utilizing 2,5-dihydroxybenzoic acid as matrix. These proteins, bovine insulin, cytochrome c, and bovine albumin have masses from approximately 5000 to 66,000 Da. Three different sample preparation techniques were investigated: aerospray, dried-drop, and acetone redeposition. Both MALDI Fourier transform and time-of-flight mass spectrometry results show that buffer tolerance of MALDI-MS samples depends upon several factors, including the relative amount of the buffer in the MALDI matrix, as well as the identity of the specific buffer. Furthermore, the rate at which buffer tolerance decreases as buffer concentration is increased varies from buffer to buffer. The current results reveal that, at very high matrix:analyte ratios, buffer tolerance of MALDI is dramatically greater than concluded in previous literature reports.

Neonatal outcomes of teenage pregnancy in a military population.

OBJECTIVE: To compare the use of prenatal care, incidence of pregnancy complications, and neonatal outcomes among adolescent mothers with those among older mothers in a military population with equal access to a tertiary care center. METHODS: We reviewed the neonatal data base of a military tertiary care center for the period 1990-1996. All singleton births at least 20 weeks' estimated gestational age (EGA) or with birth weight at least 500 g were included. Maternal transfers were excluded. Mothers were divided into five age groups, with the ranges 13-17, 18-19, 20-24, 25-29, and 30 or more years. Prenatal care was assessed by the Kessner index. Data were analyzed by chi 2 test, t test, Mann-Whitney U test, and logistic regression techniques. RESULTS: Included in the study were 10,168 infants (344 aged 13-17, 893 aged 18-19, 3244 aged 20-24, 3095 aged 25-29, and 2592 aged 30 or more years). There was a greater percentage of Hispanic mothers among adolescents aged 13-17 years (48.3%); this percentage decreased as maternal age increased (32.7% to 14.6%). The percentage of white mothers was greatest for mothers over 30 years old (72.1%), and this percentage decreased as maternal age decreased (70.3% to 35.8%). The percentage of black mothers remained relatively constant in all age groups (12.2-16.0%). There were significant differences in sponsor's rank distribution among groups; mothers 25-29 years old and over 30 years old had higher-paying ranks whereas both teenage groups were of predominantly lower-paying ranks. Gestational age at the first prenatal visit and overall use of prenatal care were significantly improved as maternal age increased for each successive age group. Older mothers had significantly higher rates for diabetes and abnormal fetal sonograms than younger mothers; younger mothers had increased rates for sexually transmitted disease, fetal growth restriction, and tobacco use. Multivariate analysis demonstrated that fetal complications, adequate prenatal care, black maternal race, and lower rank were significant independent risk factors for high-risk outcomes. Young maternal age was not an independent risk factor for prematurity or low birth weight. CONCLUSION: Although adolescent mothers (13-17 years old) used prenatal care less than older mothers, there were no differences in the incidence of prematurity, low birth weight (LBW), or neonatal survival among this group of women cared for in a military tertiary health care setting. In this health care setting, risk for premature birth and LBW are related to complications of pregnancy, maternal race, and lower-paying rank.

Erythrocyte glucose metabolism in Duchenne muscular dystrophy.

We compared glucose metabolism by erythrocytes from patients with Duchenne muscular dystrophy and by erythrocytes from control individuals. There was a significant decrease in the rate of lactate production at pH 7.2 in the dystrophic group. When the pH of the incubation medium was changed to 8.0, we found that the increase in the rate of lactate production for the dystrophic group was significantly larger. We measured the concentrations of key glycolytic metabolites and adenine nucleotides and determined the values of the energy charge during these incubations. We also determined the concentrations of polyol pathway intermediates, the activities of the oxidative portion of the pentose phosphate pathway and the activities and kinetics of phosphofructokinase from both cell groups. There were no significant differences between groups for any of these variables.

Development and evaluation of 200 novel SNP assays for population genetic studies of westslope cutthroat trout and genetic identification of related taxa.

DNA sequence data were collected and screened for single nucleotide polymorphisms (SNPs) in westslope cutthroat trout (Oncorhynchus clarki lewisi) and also for substitutions that could be used to genetically discriminate rainbow trout (O. mykiss) and cutthroat trout, as well as several cutthroat trout subspecies. In total, 260 expressed sequence tag-derived loci were sequenced and allelic discrimination genotyping assays developed from 217 of the variable sites. Another 50 putative SNPs in westslope cutthroat trout were identified by restriction-site-associated DNA sequencing, and seven of these were developed into assays. Twelve O. mykiss SNP assays that were variable within westslope cutthroat trout and 12 previously published SNP assays were also included in downstream testing. A total of 241 assays were tested on six westslope cutthroat trout populations (N = 32 per population), as well as collections of four other cutthroat trout subspecies and a population of rainbow trout. All assays were evaluated for reliability and deviation from Hardy-Weinberg and linkage equilibria. Poorly performing and duplicate assays were removed from the data set, and the remaining 200 assays were used in tests of population differentiation. The remaining markers easily distinguished the various subspecies tested, as evidenced by mean G(ST) of 0.74. A smaller subset of the markers (N = 86; average G(ST) = 0.40) was useful for distinguishing the six populations of westslope cutthroat trout. This study increases by an order of magnitude the number of genetic markers available for the study of westslope cutthroat trout and closely related taxa and includes many markers in genes (developed from ESTs).

Nutrient-rich foods: applying nutrient navigation systems to improve public health.

The American diet is high in calories, but low in nutrients. To help consumers obtain more nutrition from the calories they consume, research is underway to develop a nutrient profiling approach that can be used to evaluate individual foods and help people build healthful diets. A nutrient profiling system that rates individual foods based on their nutrient content needs to be both science-driven and user-friendly, allowing consumers to make more healthful food choices within and across all the food groups. A recent survey, commissioned by the Nutrient Rich Food Coalition, reveals that the majority of consumers and nutrition professionals believe that better information about a food's total combined nutrient content would be effective and useful in helping them make more nutrient-rich food choices.

Analysis of neutral isomeric low molecular weight carbohydrates using ferrocenyl boronate derivatization and tandem electrospray mass spectrometry.

A new analytical technique for small carbohydrates utilizing the cyclic ferrocenyl boronic esters (FcBors) of several neutral mono- and disaccharides is demonstrated. Distinction between the diastereomers of mono- and disaccharides is obtained. Analysis is by tandem electrospray-mass spectrometry (ES-MS) using a modified ion-source that promotes the preformation of ions. Selection of the molecular ion produced during single-electron oxidation of the ferrocene moiety of a specific population of saccharide isomers permits a variety of collisionally induced dissociation (CID) experiments. The resultant MS2/MS3 spectra reflect the ensemble of possible cyclic esters in equilibrium. An array of stable cross pyranose ring fragment ions representing sequential carbon loss as 30 u is observed. Consequently, the system provides an information-rich set of MSn spectra containing large amounts of structural information. Identification of D-glucose (D-Glc), its two commonly found epimers (D-mannose and D-galactose), and the two major L-diastereomers of 6-dideoxymonosaccharides, L-fucose (L-Fuc) and L-rhamnose (L-Rhm), are demonstrated. Selected pairs of disaccharides can be distinguished in terms of their anomeric linkage by reference to their MS3 spectra.

Ratiometric analysis of the ferrocene boronate esters of 2- and 4-hydroxyestradiol by tandem electrospray mass spectrometry.

A method has been developed for the semiquantitative analysis of the catechol estrogens, 2- and 4-hydroxyestradiol, using tandem electrospray mass spectrometry in a quadrupole ion trap mass analyzer. The implication of catechol estrogens in the biogenesis of breast and prostate cancer makes these labile lipophilic compounds important analytical targets. Ferrocene boronic acid is reacted with 2- and 4-hydroxyestradiol to form their cyclic boronate esters. Sample ionization is accomplished during the electrospray process by a one-electron oxidation of the ferrocene functionality to form the radical cation. The analysis depends on a non-aqueous solvent system consisting of 90% acetonitrile and 10% dichloromethane with 100 microM lithium triflate as the supporting electrolyte. The sensitivity of the analysis is greatly increased by the use of a novel electrospray interface with a large surface area stainless steel electrode coupled to a pulled fused-silica needle. Collision-induced dissociation of the selected molecular ion within the ion trap produces fragment ion spectra that can be used to distinguish between the two isobaric isomers and ultimately determine the relative amounts in mixtures containing both components. The method is sensitive to analyte concentrations in the low nM range.

Analysis of N-acetylated hexosamine monosaccharides by ferrocenyl boronation and tandem electrospray ionization mass spectrometry.

A tandem electrospray mass spectrometric (MS(n)) technique for the analysis of N-acetylated hexose carbohydrates using ferrocene boronate (F(c)Bor) derivatization was developed. The biologically important N-acetyl hexosamines can be readily distinguished utilizing this technique. The analysis is made possible by utilizing the inherent electrochemical properties of the electrospray device to produce oxidized, pre-formed single-electron ferrocenyl ions in a non-aqueous solvent system. The electrospray device was modified using a custom built cell consisting of concentric stainless steel tubes, which produced an enhanced signal for the molecular ion of each analyte species. The MS(2) spectra derived from isomeric populations of ferrocenyl boronic esters of these carbohydrates when generated under the same conditions possessed features unique to each sugar allowing easy differentiation between a number of N-acetyl hexosamine isomers.

Protein identification using a quadrupole ion trap mass spectrometer and SEQUEST database matching.

A procedure is described for rapid, sensitive protein identification utilizing liquid chromatography--tandem mass spectrometry. The analysis is performed on mixtures of peptides obtained by enzyme digestion. The SEQUEST computer program is used to match the sequence information in the spectra to a database of known protein sequences.

Introducing samples directly into electrospray ionization mass spectrometers using microscale capillary liquid chromatography.

This unit describes the design and operation of a microscale electrospray (ES) interface suitable for the on-line liquid chromatography (LC) separation and mass spectrometry (MS) analysis of mixtures of peptides and proteins. The interface utilizes an ES needle packed with reversed-phase support. Such a design has the advantage of minimizing any void volume between the end of the column and point of electrospray ionization, thus maintaining the integrity of the LC separation and maximizing sensitivity. Here, protocols are presented for construction of an integrated LC column ES needle in-house, packing the ES needle, and mounting and using the microscale ES LC/MS interface assembly. Various options for low-flow solvent delivery systems are also discussed.

Introducing samples directly into electrospray ionization mass spectrometers using a nanospray interface.

Electrospray (ES) is a concentration-dependent phenomenon, and operation at high flow rates results in a corresponding high rate of sample consumption. Sensitivity increases are most readily achieved by decreasing the flow rate of sample solution into the electrospray source. In recent years, a number of methodologies have been developed to achieve ES analyses at sub-ml/min flow rates. There are a large number of microscale ES devices currently in use, but this unit describes in detail a micro-scale interface for direct liquid introduction without separation.

Cyclic AMP in developing muscle of the rhesus monkey: effect of prostaglandin E.

Cyclic AMP levels, measured by a competitive protein-binding assay and by a prelabeling technique with adenine-14 C, were determined on fetal, infant, and adult heart, diaphragm, and skeletal muscle of the rhesus monkey (Macaca mulatta); the effect of PGE on cyclic AMP accumulation was also studied. The levels of cyclic AMP were higher in the 3 types of muscle from 150-day fetuses (91% of term) than in adult muscles and even higher at 78-100 days fetal age, and histological evidence indicated that by 65 days gestation, the majority of the rhesus skeletal muscle cells are in the myotube stage. These data agree with the observation in cell cultures that a decrease in cyclic AMP is correlated with fusion of myoblasts to form myotubes. PGE (2.8 MUM) stimulated cyclic AMP accumulation in all the fetal series, even in the earliest series (47% of term), as well as in all the adult series.

Control of glycolysis in skeletal muscle from fetal rhesus monkeys.

In our studies of metabolic control mechanisms in skeletal muscle from rhesus fetus we have determined the tissue levels of the metabolic intermediates and cofactors of the glycolytic pathway and have calculated the mass-action ratios for each reaction. Skeletal muscle from rhesus fetuses (Macaca mulatta), 90-155 days of gestational age, and from adult rhesus monkeys was used in these experiments. The apparent equilibrium constants for hexokinase and phosphofructokinase (PFK) in these tissues were over 1,000 times larger than the mass-action ratios at all ages studied; the corresponding values for pyruvate kinase were more than 800 times different. The data suggest that these three enzymes are rate-limiting for fetal skeletal muscle as early as 54% of gestation. The next step was to study some of the numerous factors that modify these non-equilibrium reactions. Increasing the ATP concentration had a marked effect on the PFK activity of both fetal and adult muscle, first increasing and then inhibiting enzyme activity. At maximum PFK activity, the amount of fructose-6-PO4(F6P) phosphorylated per mg of protein was 2-3 times greater in the two fetal than in the adult series. At a concentration of 0.3 mM, citrate decreased PFK activity of the 100-day fetal muscle; a further decrease occurred at 1.2 mM citrate. At a citrate level of 0.3 mM. the addition of inorganic phosphate (Pi) or cyclic AMP returned PFK activity to the uninhibited levels (pH 7.0). Relief of ATP inhibition of F6P phosphorylation with Pi and cyclic AMP was also observed at pH 7.0 in extracts of 100-day fetal skeletal muscle.