bluuebunny/crossref_metadata_2025_split huggingface dataset:
Green fluorescent protein–propidium iodide (GFP‐PI) based assay for flow cytometric measurement of bacterial viability
Authors: Lehtinen Janne , Nuutila Jari , Lilius Esa‐Matti 
Published: June 2004

AbstractBackgroundSeveral staining protocols have been developed for flow cytometric analysis of bacterial viability. One promising method is dual staining with the LIVE/DEAD BacLight bacterial viability kit. In this procedure, cells are treated with two different DNA‐binding dyes (SYTO9 and PI), and viability is estimated according to the proportion of bound stain. SYTO9 diffuses through the intact cell membrane and binds cellular DNA, while PI binds DNA of damaged cells only. This dual‐staining method allows effective separation between viable and dead cells, which is far more difficult to achieve with single staining. Although SYTO9‐PI dual staining is practical for various bacterial viability analyses, the method has a number of disadvantages. Specifically, the passage of SYTO9 through the cell membrane is a slow process, which is significantly accelerated when the integrity of the cell membrane is disrupted. As a result, SYTO9 binding to DNA is considerably enhanced. PI competes for binding sites with SYTO9 and may displace the bound dye. These properties diminish the reliability of the LIVE/DEAD viability kit. In this study, we investigate an alternative method for measuring bacterial viability using a combination of green fluorescent protein (GFP) and PI, with a view to improving data reliability.MethodsRecombinant Escherichia coli cells with a plasmid containing the gene for jellyfish GFP were stained with PI, and green and red fluorescence were measured by FCM. For comparison, cells containing the plasmid from which gfp was removed were stained with SYTO9 and PI, and analyzed by FCM. Viability was estimated according to the proportion of green and red fluorescence. In addition, bioluminescence and plate counting (other methods to assess viability) were used as reference procedures.ResultsSYTO9‐PI dual staining of bacterial cells revealed three different cell populations: living, compromised, and dead cells. These cell populations were more distinct when the GFP‐PI combination was used instead of dual staining. No differences in sensitivity were observed between the two methods. However, substitution of SYTO9 with GFP accelerated the procedure. Bioluminescence and plate counting results were in agreement with flow cytometric viability data.ConclusionsIn bacterial viability analyses, the GFP‐PI combination provided better distinction between current viability stages of E. coli cells than SYTO9‐PI dual staining. Additionally, the overall procedure was more rapid. No marked differences in sensitivity were observed. © 2004 Wiley‐Liss, Inc.
An analytical system based on a compact flow cytometer for DNA fragment sizing and single‐molecule detection
Authors: Habbersett Robert C. , Jett James H. 
Published: June 2004

AbstractBackgroundPrevious reports have demonstrated accurate DNA fragment sizing of linear DNA fragments, from 564 to ≈4 × 105 bp, in a flow system. B‐phycoerythrin (B‐PE), commonly used in conventional cytometric applications that require high‐sensitivity, was the first fluorophore detected in flow at the single‐molecule level.MethodsDilute solutions of stained DNA fragments or B‐PE were analyzed in a simplified, compact flow system, with enhanced performance and lower cost, utilizing a solid‐state laser and a single‐photon sensing avalanche photodiode detector (SSAPD). Extensive data processing and display software, developed specifically for the photon‐counting data stream, extracts correlated height, width, and area features from bursts of photons due to discrete molecules passing through the sensing region in the flow channel.ResultsDNA fragment sizing in flow has now been demonstrated for SYTOX‐orange–stained fragments ranging in size over 3.4 orders of magnitude, from 125 to 5 × 105 bp. For Lambda bacteriophage DNA (λ DNA; 48.5 kbp) a CV of 1.2 % has been achieved. Analysis of a femtomolar B‐PE solution demonstrates that the bursts of photons from individual molecules can be baseline‐resolved with 0.5 mW of laser power at a signal to noise ratio (SNR) of ≈30, with ≈100 photons detected from each molecule.ConclusionsA compact, low‐power, high‐sensitivity system detects DNA fragments as small as 125 bp or individual B‐PE molecules in a flowing liquid stream. Demonstrated linearity, sensitivity, and resolution indicate that <1.0 mW of laser power is optimal, permitting further miniaturization of the system and additional cost reduction. Comprehensive analytical software exploits the standard cytometric paradigm of multiple 2D graphs and gating to extract features from classes of individually analyzed biomolecules. This complete system is thus poised to engage high‐sensitivity applications not amenable to conventional flow cytometric instrumentation. © 2004 Wiley‐Liss, Inc.
The polarization of fluorescence of DNA stains depends on the incorporation density of the dye molecules
Authors: Uy Jeanne L. , Asbury Charles L. , Petersen Timothy W. , van den Engh Ger 
Published: June 2004

AbstractBackgroundThe fluorescence induced by polarized light sources, such as the lasers that are used in flow cytometry, is often polarized and anisotropic. In addition, most optical detector systems are sensitive to the direction of polarization. These two factors influence the accuracy of fluorescence intensity measurements. The intensity of two light sources can be compared only if all details of the direction and degree of polarization are known. In a previous study, we observed that fluorescence polarization might be modified by dye–dye interactions. This report further investigates the role of dye density in fluorescence polarization anisotropy.MethodsWe measured the polarization distribution of samples stained with commonly used DNA dyes. To determine the role of fluorophore proximity, we compared the monomeric and a dimeric form of the DNA dyes ethidium bromide (EB), thiazole orange (TO), and oxazole yellow (YO).ResultsIn all dyes sampled, fluorescence polarization is less at high dye concentrations than at low concentrations. The monomeric dyes exhibit a higher degree of polarization than the dimeric dyes of the same species.ConclusionsThe polarization of fluorescence from DNA dyes is related to the density of incorporation into the DNA helix. Energy transfer between molecules that are in close proximity loosens the linkage between the excitation and emission dipoles, thereby reducing the degree of polarization of the emission. © 2004 Wiley‐Liss, Inc.
Whole genome amplification for CGH analysis: Linker‐adapter PCR as the method of choice for difficult and limited samples
Authors: Pirker Christine , Raidl Maria , Steiner Elisabeth , Elbling Leonilla , Holzmann Klaus , Spiegl‐Kreinecker Sabine , Aubele Michaela , Grasl‐Kraupp Bettina , Marosi Christine , Micksche Michael , Berger Walter 
Published: June 2004

AbstractBackgroundComparative genomic hybridization (CGH) is a powerful method to investigate chromosomal imbalances in tumor cells. However, DNA quantity and quality can be limiting factors for successful CGH analysis. The aim of this study was to investigate the applicability of degenerate oligonucleotide‐primed PCR (DOP‐PCR) and a recently developed linker‐adapter‐mediated PCR (LA‐PCR) for whole genome amplification for use in CGH, especially for difficult source material.MethodsWe comparatively analyzed DNA of variable quality derived from different cell/tissue types. Additionally, dilution experiments down to the DNA content of a single cell were performed. FISH and/or classical cytogenetic analyses were used as controls.ResultsIn the case of high quality DNA samples, both methods were equally suitable for CGH. When analyzing very small amounts of these DNA samples (equivalent to one or a few human diploid cells), DOP‐PCR‐CGH, but not LA‐PCR‐CGH, frequently produced false‐positive signals (e.g., gains in 1p and 16p, and losses in chromosome 4q). In case of formalin‐fixed paraffin‐embedded tissues, success rates by LA‐PCR‐CGH were significantly higher as compared to DOP‐PCR‐CGH. DNA of minor quality frequently could be analyzed correctly by LA‐PCR‐CGH, but was prone to give false‐positive and/or false‐negative results by DOP‐PCR‐CGH.ConclusionsLA‐PCR is superior to DOP‐PCR for amplification of DNA for CGH analysis, especially in the case of very limited or partly degraded source material. © 2004 Wiley‐Liss, Inc.
Role ofSonic hedgehogin patterning of tracheal–bronchial cartilage and the peripheral lung
Authors: Miller Leigh‐Anne D. , Wert Susan E. , Clark Jean C. , Xu Yan , Perl Anne‐Karina T. , Whitsett Jeffrey A. 
Published: June 2004

AbstractSonic hedgehog(Shh) was conditionally deleted in respiratory epithelial cells of the embryonic lung in vivo. Deletion ofShhbefore embryonic day (E) 13.5 resulted in respiratory failure at birth. While lobulation was not perturbed, the lungs were hypoplastic, with reduced branching of peripheral lung tubules, evident from E13.5. Smooth muscle and endothelial cells were absent or reduced, the latter in relationship to the loss of peripheral lung parenchyma. Tracheal–bronchial ring abnormalities occurred whenShhwas deleted between E8.5 and E12.5. Deletion ofShhlater in gestation (after E13.5) caused mild abrogation of peripheral branching morphogenesis but did not disrupt tracheal‐bronchial development. Defects in branching morphogenesis and vascularization seen inShhnull mutant (Shh‐/‐) mice were substantially corrected when SHH was ectopically expressed in the respiratory epithelium; however, peripheral expression of SHH failed to correct cartilage abnormalities in the trachea and bronchi, indicating a spatial requirement for SHH expression near sites of cartilage formation. Expression of SHH by the respiratory epithelium plays an important role in the patterning of tracheal–bronchial mesenchyme required for formation of cartilage rings in conducting airways. SHH regulates branching morphogenesis and influences differentiation of the peripheral lung mesenchyme required for formation of bronchial and vascular smooth muscle. Developmental Dynamics 231:57–71, 2004. © 2004 Wiley‐Liss, Inc.
RGD‐containing peptides inhibit intestinal regeneration in the sea cucumber Holothuria glaberrima
Authors: Cabrera‐Serrano Arelys , García‐Arrarás José E. 
Published: June 2004

AbstractThe sea cucumber Holothuria glaberrima is an echinoderm capable of regenerating its viscera. Previous studies from our group have shown a striking remodeling of the extracellular matrix (ECM) during intestinal regeneration. To study the role of the ECM during regeneration, we have focused on the RGD sequences present in many ECM molecules. Regenerating animals were treated with an RGDS (Arg‐Gly‐Asp‐Ser) peptide that competes with the interaction between RGD sequence and cellular integrins. Saline and RGES (Arg‐Gly‐Glu‐Ser) peptide injections were done as controls. The size of the regenerating intestine was determined, and the regenerating structures were analyzed by immunohistochemistry for the presence of collagen and fibronectin, as well as for muscle and other cells. The results show a delay in intestinal regeneration in animals injected with the RGDS peptide, suggesting that the ECM–integrin interaction plays an important function in the regenerative process. Developmental Dynamics 231:171–178, 2004. © 2004 Wiley‐Liss, Inc.
Recapitulation of the parathyroid hormone‐related peptide–Indian hedgehog pathway in the regenerating deer antler
Authors: Faucheux C. , Nicholls B.M. , Allen S. , Danks J.A. , Horton M.A. , Price J.S. 
Published: June 2004

AbstractParathyroid hormone (PTH)‐related peptide (PTHrP) and the PTH/PTHrP receptor (PPR) play an essential role in controlling growth plate development. The aim of the present study was to use the deer antler as a model to determine whether PTHrP and PPR may also have a function in regulating cartilage and bone regeneration in an adult mammal. Antlers are the only mammalian appendages that are able to undergo repeated cycles of regeneration, and their growth from a blastema involves a modified endochondral process. Immunohistochemistry was used to establish sites of localization of PTHrP and PPR in antlers at different stages of development. The pattern of Indian Hedgehog (IHH) and transforming growth factor‐beta1 (TGFβ1) distribution was also investigated, because PTHrP expression in the developing limb is regulated by IHH and during embryonic growth plate formation TGFβ1 acts upstream of PTHrP to regulate the rate of chondrocyte differentiation. In the antler blastema (<10 days of development), PTHrP, PPR, and TGFβ1 were localized in epidermis, dermis, regenerating epithelium, and in mesenchymal cells but IHH expression was not detected. In the rapidly growing antler (weeks 4–8 of development), PTHrP, PPR, and TGF β1 were localized in skin, perichondrium, undifferentiated mesenchyme, recently differentiated chondrocytes, and in perivascular cells in cartilage but not in fully differentiated hyperytrophic chondrocytes. IHH was restricted to recently differentiated chondrocytes and to perivascular cells in cartilage. In mineralized cartilage and bone, PTHrP, PPR, IHH, and TGFβ1 were immunolocalized in perivascular cells and differentiated osteoblasts. PTHrP and PPR were also present in the periosteum. TGFβ1 in vitro stimulated PTHrP synthesis by cells from blastema, perichondrium, and cartilage. The findings of this study suggest that molecules which regulate embryonic skeletal development and postnatal epiphyseal growth may also control blastema formation, chondrogenesis, and bone formation in the regenerating deer antler. This finding is further evidence that developmental signaling pathways are recapitulated during adult mammalian bone regeneration. Developmental Dynamics 231:88–97, 2004. © 2004 Wiley‐Liss, Inc.
Modulation of activin/bone morphogenetic protein signaling by follistatin is required for the morphogenesis of mouse molar teeth
Authors: Wang Xiu‐Ping , Suomalainen Marika , Jorgez Carolina J. , Matzuk Martin M. , Wankell Miriam , Werner Sabine , Thesleff Irma 
Published: June 2004

AbstractTeeth form as ectodermal appendages, and their morphogenesis is regulated by conserved signaling pathways. The shape of the tooth crown results from growth and folding of inner dental epithelium, and the cusp patterning is regulated by transient signaling centers, the enamel knots. Several signal proteins in the transforming growth factor‐β (TGFβ) superfamily are required for tooth development. Follistatin is an extracellular inhibitor of TGFβ signaling. To investigate the roles of follistatin during tooth development, we analyzed in detail the expression patterns of follistatin, activin βA, as well asBmp2,Bmp4, andBmp7during tooth morphogenesis. We also examined the tooth phenotypes of follistatin knockout mice and of transgenic mice overexpressing follistatin in the epithelium under the keratin 14 (K14) promoter. The folding of the dental epithelium was aberrant in the molars of follistatin knockout mice, and the cusps were shallow with reduced cell proliferation and lack of anteroposterior polarization. The functions of both primary and secondary enamel knots were apparently disturbed. In K14–follistatin transgenic mice, the molar cusp pattern was also seriously affected (although different from the follistatin knockouts) and the occlusal surfaces of the molars were whorled. Their enamel was prematurely worn. In addition, all of the third molars were missing. Our results indicate that follistatin regulates morphogenesis and shaping of the tooth crown. We propose that finely tuned antagonistic effects between follistatin and TGFβ superfamily signals are critical for enamel knot formation and function, as well as for patterning of tooth cusps. Developmental Dynamics 231:98–108, 2004. © 2004 Wiley‐Liss, Inc.
A cohort‐based approach for characterizing lifetime inhalation cancer risk from time‐varying exposure to air toxics from ambient sources
Authors: Cook Richard , Jones Baxter L. , Cleland Joshua 
Published: June 2004

AbstractIn recent years, the U.S. EPA as well as state and local agencies have released a number of risk characterizations addressing potential inhalation cancer risks from exposure to ambient sources of air toxics (e.g., U.S. EPA's 1993 Motor Vehicle‐Related Air Toxics Study, the South Coast Air Quality Management District's MATES‐II study, the U.S. EPA's National Scale Air Toxics Assessment). In these assessments, quantitative estimates of cancer risk are often presented as point estimates and/or distributions of individual upper‐bound lifetime risks. The individual upper‐bound lifetime cancer risk is the probability that an individual in a given population will contract cancer if exposed continuously to a given concentration of an airborne carcinogen over an assumed lifetime. Individual risk estimates in these studies are usually based on average or median exposures for a wide population distribution.These estimates generally assume continuous exposure over an entire lifetime to levels estimated for 1 or a few years and do not account for expected changes in exposure over time. For example, risk estimates in U.S. EPA's recent National Scale Air Toxics Assessment assume individuals will be exposed to 1996 levels over a 70‐year lifetime. However, exposures to most air toxics are expected to change over time as a result of mobile and stationary source emission control programs.In this paper, a cohort‐based approach, which estimates individual lifetime cancer risk based on time‐weighted exposure over a lifetime, is presented. This approach accounts for changes in exposure level over time, including decreases anticipated as a result of regulatory programs. It also incorporates differences in exposures to children and adults that result from certain differences in time–activity patterns, integrating them into a single value for a cohort. Moreover, this paper presents a case study applying this approach to estimate average nationwide individual cancer risks from exposure to the highway mobile source contribution to ambient benzene. The case study includes estimates for the general population and a highly exposed demographic group, outdoor workers. © 2004 American Institute of Chemical Engineers Environ Prog, 23: 120–125, 2004
Case study of a potency‐weighted emission screen
Authors: Daugherty Douglas D. , Dehuff Kimberly (Mertz) , Caviness Gwen 
Published: June 2004

AbstractAccording to the U.S. Environmental Protection Agency (USEPA) document entitled Risk Assessment Guidance for Superfund (RAGS) (EPA/540/1–89/002, U.S. Environmental Protection Agency, Office of Emergency and Remedial Response, Washington, DC, 1989), a concentration‐toxicity screen is one of the chemicals of potential concern (COPCs) selection processes that may be employed to reduce the number of chemicals carried through a human health risk assessment. COPCs are those constituents for which carcinogenic risks and noncarcinogenic hazards are estimated. This paper describes the results of an analogous approach to rank chemicals at a facility according to their emission rates and potency. The objective of our proposed COPC selection methodology was to consider the comprehensive list of chemicals used in normal operations at a facility with a large chemical inventory and to identify those chemicals that have the potential to contribute at least 90 to 95% of the estimated risk for the facility (i.e., the main risk drivers). By focusing our efforts on this shorter list of chemicals, we increased the accuracy of information obtained from facility personnel and improved the level of confidence in the emission estimation, air dispersion modeling, and risk assessment results. While this approach does not consider constituent transport from multiple facility sources or proximity of receptors to those sources, these effects were evaluated in the subsequent air dispersion modeling and risk assessment of facility emissions. An evaluation of the subsequent risk assessment analyses demonstrated that the potency‐weighted emissions screen was successful in determining the list of chemicals responsible for 90 to 95% of the estimated carcinogenic risks and noncarcinogenic hazards. © 2004 American Institute of Chemical Engineers Environ Prog, 23: 168–177, 2004
Methods for incorporating uncertainty into dispersion and deposition models
Authors: Williamson Derek G. , Graettinger Andrew J. , Yegnan Anand 
Published: June 2004

AbstractDispersion and deposition models are critical components of risk assessments for airborne discharge from industrial facilities. The probabilistic nature of risk assessment, the complexities of dispersion and deposition models, and the natural variability of input parameters all support the need for probabilistic output from dispersion/deposition models. However, difficulty in implementation and data availability has prevented the routine use of uncertainty in dispersion/deposition modeling. A series of relatively simple case studies demonstrate that the Taylor series is a flexible and computationally efficient method for incorporating input uncertainty into a widely used air transport model (ISCST). Relatively simple dispersion and dispersion with deposition modeling scenarios using real input data demonstrate that the Taylor series: (1) provides similar estimates of uncertainty compared to traditional Monte Carlo simulation, (2) is far more computationally efficient, and (3) in most cases provides conservative estimates of output concentration. More over, an evaluation the computational time needed for traditional Monte Carlo simulation when considering several input variables simultaneously demonstrates the potential of the Taylor series technique to estimate uncertainty in far more complicated modeling scenarios. © 2004 American Institute of Chemical Engineers Environ Prog, 23: 158–167, 2004
Community preparedness and response model for addressing environmental risk and vulnerability
Authors: Uddin Nasim , Peters Robert W. , Haque Akhlaque 
Published: June 2004

AbstractThis paper investigates a community preparedness and response model for addressing environmental risk and vulnerability that will significantly lessen loss of human life and lower the cost of disaster recovery in a region. © 2004 American Institute of Chemical Engineers Environ Prog, 23: 126–134, 2004