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.

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.

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.

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.

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.

Biochemical and genetic analyses of the U5, U6, and U4/U6 x U5 small nuclear ribonucleoproteins from Saccharomyces cerevisiae.

We have purified the yeast U5 and U6 pre-mRNA splicing small nuclear ribonucleoproteins (snRNPs) by affinity chromatography and analyzed the associated polypeptides by mass spectrometry. The yeast U5 snRNP is composed of the two variants of U5 snRNA, six U5-specific proteins and the 7 proteins of the canonical Sm core. The U6 snRNP is composed of the U6 snRNA, Prp24, and the 7 Sm-Like (LSM) proteins. Surprisingly, the yeast DEAD-box helicase-like protein Prp28 is stably associated with the U5 snRNP, yet is absent from the purified U4/U6 x U5 snRNP. A novel yeast U5 and four novel yeast U4/U6 x U5 snRNP polypeptides were characterized by genetic and biochemical means to demonstrate their involvement in the pre-mRNA splicing reaction. We also show that, unlike the human tri-snRNP, the yeast tri-snRNP dissociated upon addition of ATP or dATP.

p22 is a novel plasminogen fragment with antiangiogenic activity.

Tumor or tumor-associated cells cleave circulating plasminogen into three or four kringle-containing antiangiogenic fragments, collectively referred to as angiostatin. Angiostatin blocks tumor growth and metastasis by preventing the growth of endothelial cells that are critical for tumor vascularization. Here, we show that cancer and normal cells convert plasminogen into a novel 22 kDa fragment (p22). Production of this plasminogen fragment in a cell-free system has allowed characterization of the structure and activity of the protein. p22 consists of amino acid residues 78-180 of plasminogen and therefore embodies the first plasminogen kringle (residues 84-162) as well as additional N- and C-terminal residues. Circular dichroism and intrinsic fluorescence spectrum analysis have defined structural differences between p22 and recombinant plasminogen kringle 1 (rK1), therefore suggesting a unique conformation for kringle 1 within p22. Proliferation of capillary endothelial cells but not cells of other lineages was selectively inhibited by p22 in vitro. In addition, p22 prevented vascular growth of chick chorioallantoic membranes (CAMs) in vivo. Furthermore, administration of p22 at low dose suppressed the growth of murine Lewis lung carcinoma (LLC) metastatic foci in vivo. This is the first identification of a single kringle-containing antiangiogenic plasminogen fragment produced under physiological conditions.

Transcription of chromosomal rRNA genes by both RNA polymerase I and II in yeast uaf30 mutants lacking the 30 kDa subunit of transcription factor UAF.

UAF, a yeast RNA polymerase I transcription factor, contains Rrn5p, Rrn9p, Rrn10p, histones H3 and H4, and uncharacterized protein p30. Mutants defective in RRN5, RRN9 or RRN10 are unable to transcribe rDNA by polymerase I and grow extremely slowly, but give rise to variants able to grow by transcribing chromosomal rDNA by polymerase II. Thus, UAF functions as both an activator of polymerase I and a silencer of polymerase II for rDNA transcription. We have now identified the gene for subunit p30. This gene, UAF30, is not essential for growth, but its deletion decreases the cellular growth rate. Remarkably, the deletion mutants use both polymerase I and II for rDNA transcription, indicating that the silencer function of UAF is impaired, even though rDNA transcription by polymerase I is still occurring. A UAF complex isolated from the uaf30 deletion mutant was found to retain the in vitro polymerase I activator function to a large extent. Thus, Uaf30p plays only a minor role in its activator function. Possible reasons for slow growth caused by uaf30 mutations are discussed.

Osteoclasts secrete the chemotactic cytokine mim-1.

Osteoclasts are terminally differentiated, multinucleated cells of monocytic origin. In this study, we report that osteoclasts secrete a 35 kD protein and that phorbol myristate acetate treatment stimulates secretion dramatically. Peptide digests of the protein were analyzed by mass spectroscopy. The protein was identified as myb induced myeloid protein-1 precursor (mim-1 protein). Mim-1 is expressed specifically by hematopoietic cells and has no known function. It is homologous with the neutrophil chemokine, chondromodulin II, which stimulates proliferation of osteoblasts and chondrocytes. Western analysis showed that osteoclasts secrete mim-1 into culture media. Immunofluorescence studies demonstrated a cytoplasmic and perinuclear distribution of mim-1 in both avian osteoclasts and human osteoclast-like cells. Expression and secretion of a chemokine-like protein by osteoclasts suggests a novel signaling pathway in the bone microenvironment that may be involved in coordinating bone remodeling.

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.

Purification and characterization of A61. An angiostatin-like plasminogen fragment produced by plasmin autodigestion in the absence of sulfhydryl donors.

Plasmin, a broad spectrum proteinase, is inactivated by an autoproteolytic reaction that results in the destruction of the heavy and light chains of the protein. Recently we demonstrated that a 61-kDa plasmin fragment was one of the major products of this autoproteolytic reaction (Fitzpatrick, S. L., Kassam, G., Choi, K. S., Kang, H. M., Fogg, D. K., and Waisman, D. M. (2000)Biochemistry 39, 1021-1028). In the present communication we have identified the 61-kDa plasmin fragment as a novel four kringle-containing protein consisting of the amino acid sequence Lys(78)-Lys(468). To avoid confusion with the plasmin(ogen) fragment, angiostatin(R) (Lys(78)-Ala(440)), we have named this protein A(61). Unlike angiostatin, A(61) was produced in vitro from plasmin autodigestion in the absence of sulfhydryl donors. A(61) bound to lysine-Sepharose and also underwent a large increase in fluorescence yield upon binding of the lysine analogue, trans-4-aminomethylcyclohexanecarboxylic acid. Circular dichroism suggested that A(61) was composed of 21% beta-strand, 14% beta-turn, 18% 3(1)-helix and 8% 3(10)-helix. A(61) was an anti-angiogenic protein as indicated by the inhibition of bovine capillary endothelial cell proliferation. Plasminogen was converted to A(61) by HT1080 cells and bovine capillary endothelial cells. Furthermore, a plasminogen fragment similar to A(61) was present in the serum of humans as well as normal and tumor-bearing mice. These results establish that plasmin turnover can generate anti-angiogenic plasmin fragments in a nonpathological setting.

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.

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.

The human SWI/SNF-B chromatin-remodeling complex is related to yeast rsc and localizes at kinetochores of mitotic chromosomes.

The SWI/SNF family of chromatin-remodeling complexes facilitates gene expression by helping transcription factors gain access to their targets in chromatin. SWI/SNF and Rsc are distinctive members of this family from yeast. They have similar protein components and catalytic activities but differ in biological function. Rsc is required for cell cycle progression through mitosis, whereas SWI/SNF is not. Human complexes of this family have also been identified, which have often been considered related to yeast SWI/SNF. However, all human subunits identified to date are equally similar to components of both SWI/SNF and Rsc, leaving open the possibility that some or all of the human complexes are rather related to Rsc. Here, we present evidence that the previously identified human SWI/SNF-B complex is indeed of the Rsc type. It contains six components conserved in both Rsc and SWI/SNF. Importantly, it has a unique subunit, BAF180, that harbors a distinctive set of structural motifs characteristic of three components of Rsc. Of the two mammalian ATPases known to be related to those in the yeast complexes, human SWI/SNF-B contains only the homolog that functions like Rsc during cell growth. Immunofluorescence studies with a BAF180 antibody revealed that SWI/SNF-B localizes at the kinetochores of chromosomes during mitosis. Our data suggest that SWI/SNF-B and Rsc represent a novel subfamily of chromatin-remodeling complexes conserved from yeast to human, and could participate in cell division at kinetochores of mitotic chromosomes.

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.

Oligosaccharide profiling: the facile detection of mono-, di- and oligosaccharides by electrospray orthogonal time-of-flight mass spectrometry using 3-aminophenylboronic acid derivatization.

Monosaccharides, disaccharides and larger carbohydrates can be derivatized using 3-aminophenylboronic acid (3-APBA). This procedure is carried out at low pH (2.7-3.0) and allows the use of positive ion mode electrospray orthogonal time-of-flight mass spectrometry (ES-OTOFMS) to analyze the resulting boronate complexes. A carbohydrate profile map of a complex carbohydrate mixture, honey, was prepared which displayed superior sensitivity when compared with lithium ion cationization. Complexes formed using simple mono- and disaccharides show that facile in situ derivatization leads to an equilibrium mixture; which is reproducible for a specific set of electrospray conditions. D-Glucose could be detected at 5 microM concentration using the standard instrument spray interface. Lower detection levels of approximately 500 nM could be achieved using a nanospray device. The 3-APBA complexes are observed on instruments employing a low temperature interface (140-150 degrees C) which allows formation of the boronate species while still promoting efficient desolvation of the ions. The spectral identification of 3-APBA complexed carbohydrates in complex mixtures is facilitated by the easily observed 1 mass unit separated peak pair bearing the 1:4 ratio resulting from the natural isotopic abundance of (10)B and (11)B.

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.