Publications by authors named "Daniel Watkins"

33 Publications

Dynamics of Membrane Proteins Monitored by Single-Molecule Fluorescence Across Multiple Timescales.

Methods Mol Biol 2020 ;2168:273-297

Faculty of Science, University of South Bohemia, Ceske Budejovice, Czech Republic.

Single-molecule techniques provide insights into the heterogeneity and dynamics of ensembles and enable the extraction of mechanistic information that is complementary to high-resolution structural techniques. Here, we describe the application of single-molecule Förster resonance energy transfer to study the dynamics of integral membrane protein complexes on timescales spanning sub-milliseconds to minutes (10-10 s).
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http://dx.doi.org/10.1007/978-1-0716-0724-4_13DOI Listing
March 2021

Association of Fluorescent Protein Pairs and Its Significant Impact on Fluorescence and Energy Transfer.

Adv Sci (Weinh) 2020 Jan 23;8(1):2003167. Epub 2020 Nov 23.

Molecular Biosciences School of Biosciences Cardiff University Cardiff CF10 3AX UK.

Fluorescent proteins (FPs) are commonly used in pairs to monitor dynamic biomolecular events through changes in proximity via distance dependent processes such as Förster resonance energy transfer (FRET). The impact of FP association is assessed by predicting dimerization sites in silico and stabilizing the dimers by bio-orthogonal covalent linkages. In each tested case dimerization changes inherent fluorescence, including FRET. GFP homodimers demonstrate synergistic behavior with the dimer being brighter than the sum of the monomers. The homodimer structure reveals the chromophores are close with favorable transition dipole alignments and a highly solvated interface. Heterodimerization (GFP with Venus) results in a complex with ≈87% FRET efficiency, significantly below the 99.7% efficiency predicted. A similar efficiency is observed when the wild-type FPs are fused to a naturally occurring protein-protein interface system. GFP complexation with mCherry results in loss of mCherry fluorescence. Thus, simple assumptions used when monitoring interactions between proteins via FP FRET may not always hold true, especially under conditions whereby the protein-protein interactions promote FP interaction.
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http://dx.doi.org/10.1002/advs.202003167DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7788595PMC
January 2020

Refined measurement of SecA-driven protein secretion reveals that translocation is indirectly coupled to ATP turnover.

Proc Natl Acad Sci U S A 2020 12 30;117(50):31808-31816. Epub 2020 Nov 30.

School of Biochemistry, University of Bristol, BS8 1TD Bristol, United Kingdom

The universally conserved Sec system is the primary method cells utilize to transport proteins across membranes. Until recently, measuring the activity-a prerequisite for understanding how biological systems work-has been limited to discontinuous protein transport assays with poor time resolution or reported by large, nonnatural tags that perturb the process. The development of an assay based on a split superbright luciferase (NanoLuc) changed this. Here, we exploit this technology to unpick the steps that constitute posttranslational protein transport in bacteria. Under the conditions deployed, the transport of a model preprotein substrate (proSpy) occurs at 200 amino acids (aa) per minute, with SecA able to dissociate and rebind during transport. Prior to that, there is no evidence for a distinct, rate-limiting initiation event. Kinetic modeling suggests that SecA-driven transport activity is best described by a series of large (∼30 aa) steps, each coupled to hundreds of ATP hydrolysis events. The features we describe are consistent with a nondeterministic motor mechanism, such as a Brownian ratchet.
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http://dx.doi.org/10.1073/pnas.2010906117DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7749344PMC
December 2020

Controlling Protein Nanocage Assembly with Hydrostatic Pressure.

J Am Chem Soc 2020 12 30;142(49):20640-20650. Epub 2020 Nov 30.

School of Cellular and Molecular Medicine, University of Bristol, University Walk, Bristol BS8 1TD, U.K.

Controlling the assembly and disassembly of nanoscale protein cages for the capture and internalization of protein or non-proteinaceous components is fundamentally important to a diverse range of bionanotechnological applications. Here, we study the reversible, pressure-induced dissociation of a natural protein nanocage, bacterioferritin (Bfr), using synchrotron radiation small-angle X-ray scattering (SAXS) and circular dichroism (CD). We demonstrate that hydrostatic pressures of 450 MPa are sufficient to completely dissociate the Bfr 24-mer into protein dimers, and the reversibility and kinetics of the reassembly process can be controlled by selecting appropriate buffer conditions. We also demonstrate that the heme B prosthetic group present at the subunit dimer interface influences the stability and pressure lability of the cage, despite its location being discrete from the interdimer interface that is key to cage assembly. This indicates a major cage-stabilizing role for heme within this family of ferritins.
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http://dx.doi.org/10.1021/jacs.0c07285DOI Listing
December 2020

Multi-stimulus linear negative expansion of a breathing M(OCR)-node MOF.

Faraday Discuss 2021 Feb 12;225:133-151. Epub 2020 Nov 12.

Department of Chemistry, University of Sheffield, Brook Hill, Sheffield S3 7HF, UK.

The metal-organic framework (MeNH)[Cd(NOBDC)] (SHF-81) comprises flattened tetrahedral Cd(OCR) nodes, in which Cd(ii) centres are linked via NOBDC ligands (2-nitrobenzene-1,4-dicarboxylate) to give a doubly interpenetrated anionic network, with charge balanced by two MeNH cations per Cd centre resident in the pores. The study establishes that this is a twinned α-quartz-type structure (trigonal, space group P321, x = 1 or 2), although very close to the higher symmetry β-quartz arrangement (hexagonal, P622, x = 2 or 4) in its as-synthesised solvated form [Cd(NOBDC)]·2DMF·0.5HO (SHF-81-DMF). The activated MOF exhibits very little N uptake at 77 K, but shows significant CO uptake at 273-298 K with an isosteric enthalpy of adsorption (ΔH) at zero coverage of -27.4 kJ mol determined for the MOF directly activated from SHF-81-DMF. A series of in situ diffraction experiments, both single-crystal X-ray diffraction (SCXRD) and powder X-ray diffraction (PXRD), reveal that the MOF is flexible and exhibits breathing behaviour with observed changes as large as 12% in the a- and b-axes (|Δa|, |Δb| < 1.8 Å) and 5.5% in the c-axis (|Δc| < 0.7 Å). Both the solvated SHF-81-DMF and activated/desolvated SHF-81 forms of the MOF exhibit linear negative thermal expansion (NTE), in which pores that run parallel to the c-axis expand in diameter (a- and b-axis) while contracting in length (c-axis) upon increasing temperature. Adsorption of CO gas at 298 K also results in linear negative expansion (Δa, Δb > 0; Δc < 0; ΔV > 0). The largest change in dimensions is observed during activation/desolvation from SHF-81-DMF to SHF-81 (Δa, Δb < 0; Δc > 0; ΔV < 0). Collectively the nine in situ diffraction experiments conducted suggest the breathing behaviour is continuous, although individual desolvation and adsorption experiments do not rule out the possibility of a gating or step at intermediate geometries that is coupled with continuous dynamic behaviour towards the extremities of the breathing amplitude.
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http://dx.doi.org/10.1039/d0fd00089bDOI Listing
February 2021

Inter-membrane association of the Sec and BAM translocons for bacterial outer-membrane biogenesis.

Elife 2020 11 4;9. Epub 2020 Nov 4.

School of Biochemistry, University of Bristol, Bristol, United Kingdom.

The outer-membrane of Gram-negative bacteria is critical for surface adhesion, pathogenicity, antibiotic resistance and survival. The major constituent - hydrophobic β-barrel uter-embrane roteins (OMPs) - are first secreted across the inner-membrane through the Sec-translocon for delivery to periplasmic chaperones, for example SurA, which prevent aggregation. OMPs are then offloaded to the β-arrel ssembly achinery (BAM) in the outer-membrane for insertion and folding. We show the olo-ransocon (HTL) - an assembly of the protein-channel core-complex SecYEG, the ancillary sub-complex SecDF, and the membrane 'insertase' YidC - contacts BAM through periplasmic domains of SecDF and YidC, ensuring efficient OMP maturation. Furthermore, the proton-motive force (PMF) across the inner-membrane acts at distinct stages of protein secretion: (1) SecA-driven translocation through SecYEG and (2) communication of conformational changes via SecDF across the periplasm to BAM. The latter presumably drives efficient passage of OMPs. These interactions provide insights of inter-membrane organisation and communication, the importance of which is becoming increasingly apparent.
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http://dx.doi.org/10.7554/eLife.60669DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7695460PMC
November 2020

A High-Resolution Luminescent Assay for Rapid and Continuous Monitoring of Protein Translocation across Biological Membranes.

J Mol Biol 2019 04 13;431(8):1689-1699. Epub 2019 Mar 13.

School of Biochemistry, University of Bristol, Bristol, UK; BrisSynBio, University of Bristol, Bristol, UK. Electronic address:

Protein translocation is a fundamental process in biology. Major gaps in our understanding of this process arise due the poor sensitivity, low time resolution and irreproducibility of translocation assays. To address this, we applied NanoLuc split-luciferase to produce a new strategy for measuring protein transport. The system reduces the timescale of data collection from days to minutes and allows for continuous acquisition with a time resolution in the order of seconds, yielding kinetics parameters suitable for mechanistic elucidation and mathematical fitting. To demonstrate its versatility, we implemented and validated the assay in vitro and in vivo for the bacterial Sec system and the mitochondrial protein import apparatus. Overall, this technology represents a major step forward, providing a powerful new tool for fundamental mechanistic enquiry of protein translocation and for inhibitor (drug) screening, with an intensity and rigor unattainable through classical methods.
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http://dx.doi.org/10.1016/j.jmb.2019.03.007DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6461198PMC
April 2019

Specific cardiolipin-SecY interactions are required for proton-motive force stimulation of protein secretion.

Proc Natl Acad Sci U S A 2018 07 16;115(31):7967-7972. Epub 2018 Jul 16.

School of Biochemistry, University of Bristol, Bristol BS8 1TD, United Kingdom;

The transport of proteins across or into membranes is a vital biological process, achieved in every cell by the conserved Sec machinery. In bacteria, SecYEG combines with the SecA motor protein for secretion of preproteins across the plasma membrane, powered by ATP hydrolysis and the transmembrane proton-motive force (PMF). The activities of SecYEG and SecA are modulated by membrane lipids, particularly cardiolipin (CL), a specialized phospholipid known to associate with a range of energy-transducing machines. Here, we identify two specific CL binding sites on the SecA-SecYEG complex, through application of coarse-grained molecular dynamics simulations. We validate the computational data and demonstrate the conserved nature of the binding sites using in vitro mutagenesis, native mass spectrometry, biochemical analysis, and fluorescence spectroscopy of SecYEG. The results show that the two sites account for the preponderance of functional CL binding to SecYEG, and mediate its roles in ATPase and protein transport activity. In addition, we demonstrate an important role for CL in the conferral of PMF stimulation of protein transport. The apparent transient nature of the CL interaction might facilitate proton exchange with the Sec machinery, and thereby stimulate protein transport, by a hitherto unexplored mechanism. This study demonstrates the power of coupling the high predictive ability of coarse-grained simulation with experimental analyses, toward investigation of both the nature and functional implications of protein-lipid interactions.
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http://dx.doi.org/10.1073/pnas.1721536115DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6077702PMC
July 2018

Dynamic action of the Sec machinery during initiation, protein translocation and termination.

Elife 2018 06 7;7. Epub 2018 Jun 7.

Astbury Centre for Structural Molecular Biology, University of Leeds, Leeds, United Kingdom.

Protein translocation across cell membranes is a ubiquitous process required for protein secretion and membrane protein insertion. In bacteria, this is mostly mediated by the conserved SecYEG complex, driven through rounds of ATP hydrolysis by the cytoplasmic SecA, and the trans-membrane proton motive force. We have used single molecule techniques to explore SecY pore dynamics on multiple timescales in order to dissect the complex reaction pathway. The results show that SecA, both the signal sequence and mature components of the pre-protein, and ATP hydrolysis each have important and specific roles in channel unlocking, opening and priming for transport. After channel opening, translocation proceeds in two phases: a slow phase independent of substrate length, and a length-dependent transport phase with an intrinsic translocation rate of ~40 amino acids per second for the proOmpA substrate. Broad translocation rate distributions reflect the stochastic nature of polypeptide transport.
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http://dx.doi.org/10.7554/eLife.35112DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6021171PMC
June 2018

Site-Specific One-to-One Click Coupling of Single Proteins to Individual Carbon Nanotubes: A Single-Molecule Approach.

J Am Chem Soc 2017 12 29;139(49):17834-17840. Epub 2017 Nov 29.

School of Biological and Chemical Sciences, Institute of Bioengineering, and Materials Research Institute, Queen Mary University of London , Mile End Road, London E1 4NS, United Kingdom.

We report the site-specific coupling of single proteins to individual carbon nanotubes (CNTs) in solution and with single-molecule control. Using an orthogonal Click reaction, Green Fluorescent Protein (GFP) was engineered to contain a genetically encoded azide group and then bound to CNT ends in different configurations: in close proximity or at longer distances from the GFP's functional center. Atomic force microscopy and fluorescence analysis in solution and on surfaces at the single-protein level confirmed the importance of bioengineering optimal protein attachment sites to achieve direct protein-nanotube communication and bridging.
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http://dx.doi.org/10.1021/jacs.7b07362DOI Listing
December 2017

Construction and in vivo assembly of a catalytically proficient and hyperthermostable de novo enzyme.

Nat Commun 2017 08 25;8(1):358. Epub 2017 Aug 25.

School of Biochemistry, University of Bristol, University Walk, Bristol, BS8 1TD, UK.

Although catalytic mechanisms in natural enzymes are well understood, achieving the diverse palette of reaction chemistries in re-engineered native proteins has proved challenging. Wholesale modification of natural enzymes is potentially compromised by their intrinsic complexity, which often obscures the underlying principles governing biocatalytic efficiency. The maquette approach can circumvent this complexity by combining a robust de novo designed chassis with a design process that avoids atomistic mimicry of natural proteins. Here, we apply this method to the construction of a highly efficient, promiscuous, and thermostable artificial enzyme that catalyzes a diverse array of substrate oxidations coupled to the reduction of HO. The maquette exhibits kinetics that match and even surpass those of certain natural peroxidases, retains its activity at elevated temperature and in the presence of organic solvents, and provides a simple platform for interrogating catalytic intermediates common to natural heme-containing enzymes.Catalytic mechanisms of enzymes are well understood, but achieving diverse reaction chemistries in re-engineered proteins can be difficult. Here the authors show a highly efficient and thermostable artificial enzyme that catalyzes a diverse array of substrate oxidations coupled to the reduction of HO.
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http://dx.doi.org/10.1038/s41467-017-00541-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5572459PMC
August 2017

Effect of Bioconjugation on the Reduction Potential of Heme Proteins.

Biomacromolecules 2016 11 3;17(11):3485-3492. Epub 2016 Oct 3.

School of Chemistry, University of Bristol , Bristol BS8 1TS, United Kingdom.

The modification of protein surfaces employing cationic and anionic species enables the assembly of these biomaterials into highly sophisticated hierarchical structures. Such modifications can allow bioconjugates to retain or amplify their functionalities under conditions in which their native structure would be severely compromised. In this work, we assess the effect of this type of bioconjugation on the redox properties of two model heme proteins, that is, cytochrome c (CytC) and myoglobin (Mb). In particular, the work focuses on the sequential modification by 3-dimethylamino propylamine (DMAPA) and 4-nonylphenyl 3-sulfopropyl ether (S1) anionic surfactant. Bioconjugation with DMAPA and S1 are the initial steps in the generation of pure liquid proteins, which remain active in the absence of water and up to temperatures above 150 °C. Thin-layer spectroelectrochemistry reveals that DMAPA cationization leads to a distribution of bioconjugate structures featuring reduction potentials shifted up to 380 mV more negative than the native proteins. Analysis based on circular dichroism, MALDI-TOF mass spectrometry, and zeta potential measurements suggest that the shift in the reduction potentials are not linked to protein denaturation, but to changes in the spin state of the heme. These alterations of the spin states originate from subtle structural changes induced by DMAPA attachment. Interestingly, electrostatic coupling of anionic surfactant S1 shifts the reduction potential closer to that of the native protein, demonstrating that the modifications of the heme electronic configuration are linked to surface charges.
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http://dx.doi.org/10.1021/acs.biomac.6b00928DOI Listing
November 2016

Patients exposed to diagnostic head and neck radiation for the management of shunted hydrocephalus have a significant risk of developing thyroid nodules.

Pediatr Surg Int 2016 Jun 15;32(6):565-9. Epub 2016 Apr 15.

Division of Pediatric Surgery, Department of Surgery, The Ohio State University College of Medicine, Nationwide Children's Hospital, 700 Children's Drive, ED320, Columbus, OH, 43205, USA.

Purpose: External radiation to the head and neck can lead to an increased incidence of thyroid nodules. We investigated whether patients requiring repeated head and neck imaging for the management of shunted hydrocephalus had a higher incidence of ultrasound-detected thyroid nodules compared to reports of comparable age.

Methods: Patients treated at our institution for shunted hydrocephalus from 1990 to 2003 were contacted. Enroled patients underwent a thyroid ultrasound. Demographic data and radiation exposure history were obtained retrospectively.

Results: Thyroid nodules were identified sonographically in 15/112 patients (13.6 %). Patients with thyroid nodules were older (mean 24.3 ± 7.6 years) than those without (mean 18.4 ± 8.0 years) (p = 0.005). Those with a detectable thyroid nodule had a longer follow up time compared to those who did not (mean 21.9 ± 5.5 vs. 15.1 ± 7 years, respectively) (p = 0.018).

Conclusion: Patients with shunted hydrocephalus are exposed to substantial head and neck radiation from diagnostic imaging and have a higher incidence of thyroid nodules detected by ultrasonography. These patients should be provided ongoing surveillance for detection of thyroid nodules and the possibility of malignancy.
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http://dx.doi.org/10.1007/s00383-016-3894-1DOI Listing
June 2016

A suite of de novo c-type cytochromes for functional oxidoreductase engineering.

Biochim Biophys Acta 2016 May 10;1857(5):493-502. Epub 2015 Nov 10.

School of Biochemistry, University of Bristol, University Walk, Bristol BS8 1TD, UK. Electronic address:

Central to the design of an efficient de novo enzyme is a robust yet mutable protein scaffold. The maquette approach to protein design offers precisely this, employing simple four-α-helix bundle scaffolds devoid of evolutionary complexity and with proven tolerance towards iterative protein engineering. We recently described the design of C2, a de novo designed c-type cytochrome maquette that undergoes post-translational modification in E. coli to covalently graft heme onto the protein backbone in vivo. This de novo cytochrome is capable of reversible oxygen binding, an obligate step in the catalytic cycle of many oxygen-activating oxidoreductases. Here we demonstrate the flexibility of both the maquette platform and the post-translational machinery of E. coli by creating a suite of functional de novo designed c-type cytochromes. We explore the engineering tolerances of the maquette by selecting alternative binding sites for heme C attachment and creating di-heme maquettes either by appending an additional heme C binding motif to the maquette scaffold or by binding heme B through simple bis-histidine ligation to a second binding site. The new designs retain the essential properties of the parent design but with significant improvements in structural stability. Molecular dynamics simulations aid the rationalization of these functional improvements while providing insight into the rules for engineering heme C binding sites in future iterations. This versatile, functional suite of de novo c-type cytochromes shows significant promise in providing robust platforms for the future engineering of de novo oxygen-activating oxidoreductases. This article is part of a Special Issue entitled Biodesign for Bioenergetics--the design and engineering of electron transfer cofactors, proteins and protein networks, edited by Ronald L. Koder and J.L. Ross Anderson.
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http://dx.doi.org/10.1016/j.bbabio.2015.11.003DOI Listing
May 2016

An unusual case of foreign body pulmonary embolus: case report and review of penetrating trauma at a pediatric trauma center.

Pediatr Surg Int 2015 Mar 30;31(3):241-7. Epub 2015 Jan 30.

Department of Pediatric Surgery, Nationwide Children's Hospital, 700 Children's Drive, ED 383, Columbus, OH, 43205, USA.

Purpose: Penetrating thoracic trauma is relatively rare in the pediatric population. Embolization of foreign bodies from penetrating trauma is very uncommon. We present a case of a 6-year-old boy with a penetrating foreign body from a projectile dislodged from a lawn mower. Imaging demonstrated a foreign body that embolized to the left pulmonary artery, which was successfully treated non-operatively.

Methods: We reviewed the penetrating thoracic trauma patients in the trauma registry at our institution between 1/1/03 and 12/31/12. Data collected included demographic data, procedures performed, complications and outcome.

Results: Sixty-five patients were identified with a diagnosis of penetrating thoracic trauma. Fourteen of the patients had low velocity penetrating trauma and 51 had high velocity injuries. Patients with high velocity injuries were more likely to be older and less likely to be Caucasian. There were no statistically significant differences between patients with low vs. high velocity injuries regarding severity scores or length of stay. There were no statistically significant differences in procedures required between patients with low and high velocity injuries.

Conclusions: Penetrating thoracic trauma is rare in children. The case presented here represents the only report of cardiac foreign body embolus we could identify in a pediatric patient.
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http://dx.doi.org/10.1007/s00383-015-3659-2DOI Listing
March 2015

HB-EGF augments the ability of mesenchymal stem cells to attenuate intestinal injury.

J Pediatr Surg 2014 Jun 31;49(6):938-44; discussion 944. Epub 2014 Jan 31.

The Research Institute at Nationwide Children's Hospital, Center for Perinatal Research, Department of Pediatric Surgery, Nationwide Children's Hospital, and The Ohio State University College of Medicine, Columbus, Ohio. Electronic address:

Background: We have previously demonstrated that heparin-binding EGF-like growth factor (HB-EGF) and mesenchymal stem cell (MSC) administration protect the intestines from ischemia/reperfusion (I/R) injury in vivo, with amniotic fluid-derived MSC (AF-MSC) being more efficacious than bone marrow-derived MSC (BM-MSC). The goal of the current study was to determine whether the protective effects of HB-EGF were from direct effects on MSC or via alternative mechanisms.

Methods: Murine MSC were transfected with an HB-EGF plasmid or control plasmid by electroporation. Mice were subjected to segmental intestinal I/R injury and received either BM-MSC or AF-MSC either with or without exogenous HB-EGF, or BM-MSC or AF-MSC that endogenously over-expressed HB-EGF. MSC engraftment, intestinal histologic injury, and intestinal permeability were quantified.

Results: There was increased MSC engraftment into injured compared to uninjured intestine. HB-EGF increased AF-MSC engraftment into injured intestine. Administration of HB-EGF and MSC improved intestinal histology and intestinal permeability after I/R injury, with AF-MSC being most efficacious. The effect of HB-EGF on MSC was similar when the growth factor was administered exogenously, or when it was overexpressed endogenously.

Conclusions: The effect of HB-EGF on AF-MSC was similar with both exogenous administration and endogenous overexpression of the growth factor, implying that HB-EGF has a direct effect on AF-MSC. This information may assist in guiding potential future AF-MSC-based therapies for patients at risk of intestinal ischemic injuries.
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http://dx.doi.org/10.1016/j.jpedsurg.2014.01.030DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4044538PMC
June 2014

HMGB1-promoted and TLR2/4-dependent NK cell maturation and activation take part in rotavirus-induced murine biliary atresia.

PLoS Pathog 2014 Mar 20;10(3):e1004011. Epub 2014 Mar 20.

Department of Pediatric Surgery, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China.

Recent studies show that NK cells play important roles in murine biliary atresia (BA), and a temporary immunological gap exists in this disease. In this study, we found high-mobility group box-1 (HMGB1) and TLRs were overexpressed in human and rotavirus-induced murine BA. The overexpressed HMGB1 released from the nuclei of rotavirus-infected cholangiocytes, as well as macrophages, activated hepatic NK cells via HMGB1-TLRs-MAPK signaling pathways. Immature NK cells had low cytotoxicity on rotavirus-injured cholangiocytes due to low expression of TLRs, which caused persistent rotavirus infection in bile ducts. HMGB1 up-regulated the levels of TLRs of NK cells and promoted NK cell activation in an age-dependent fashion. As NK cells gained increasing activation as mice aged, they gained increasing cytotoxicity on rotavirus-infected cholangiocytes, which finally caused BA. Adult NK cells eliminated rotavirus-infected cholangiocytes shortly after infection, which prevented persistent rotavirus infection in bile ducts. Moreover, adoptive transfer of mature NK cells prior to rotavirus infection decreased the incidence of BA in newborn mice. Thus, the dysfunction of newborn NK cells may, in part, participate in the immunological gap in the development of rotavirus induced murine BA.
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http://dx.doi.org/10.1371/journal.ppat.1004011DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3961347PMC
March 2014

Constructing a man-made c-type cytochrome maquette : electron transfer, oxygen transport and conversion to a photoactive light harvesting maquette.

Chem Sci 2014 Feb 31;5(2):507-514. Epub 2013 Oct 31.

The Johnson Research Foundation, Dept. of Biochemistry and Biophysics, University of Pennsylvania, PA19104-6059, USA.

The successful use of man-made proteins to advance synthetic biology requires both the fabrication of functional artificial proteins in a living environment, and the ability of these proteins to interact productively with other proteins and substrates in that environment. Proteins made by the maquette method integrate sophisticated oxidoreductase function into evolutionarily naive, non-computationally designed protein constructs with sequences that are entirely unrelated to any natural protein. Nevertheless, we show here that we can efficiently interface with the natural cellular machinery that covalently incorporates heme into natural cytochromes c to produce an artificial c-type cytochrome maquette. Furthermore, this c-type cytochrome maquette is designed with a displaceable histidine heme ligand that opens to allow functional oxygen binding, the primary event in more sophisticated functions ranging from oxygen storage and transport to catalytic hydroxylation. To exploit the range of functions that comes from the freedom to bind a variety of redox cofactors within a single maquette framework, this c-type cytochrome maquette is designed with a second, non-heme C, tetrapyrrole binding site, enabling the construction of an elementary electron transport chain, and when the heme C iron is replaced with zinc to create a Zn porphyrin, a light-activatable artificial redox protein. The work we describe here represents a major advance in protein design, offering a robust platform for new c-type heme based oxidoreductase designs and an equally important proof-of-principle that cofactor-equipped man-made proteins can be expressed in living cells, paving the way for constructing functionally useful man-made proteins .
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http://dx.doi.org/10.1039/C3SC52019FDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3952003PMC
February 2014

De novo protein components for oxidoreductase assembly and biological integration.

Curr Opin Chem Biol 2014 Apr 5;19:90-8. Epub 2014 Mar 5.

School of Biochemistry, University of Bristol, University Walk, Bristol BS8 1TD, UK. Electronic address:

Manmade protein design is founded on the concept that a protein with minimal evolutionary complexity is a viable scaffold for incorporating simple engineering elements responsible for function in natural proteins and enzymes. There has been significant, recent success both in fabricating manmade protein components that exhibit functional elements inspired by natural oxidoreductases, and the functional integration of this componentry with natural proteins and biochemical pathways. Here we discuss the state of the art in de novo oxidoreductase construction, focusing on the diverse manmade componentry available and how their functions might be interfaced and integrated within living organisms.
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http://dx.doi.org/10.1016/j.cbpa.2014.01.016DOI Listing
April 2014

Enteric nervous system abnormalities are present in human necrotizing enterocolitis: potential neurotransplantation therapy.

Stem Cell Res Ther 2013 ;4(6):157

Introduction: Intestinal dysmotility following human necrotizing enterocolitis suggests that the enteric nervous system is injured during the disease. We examined human intestinal specimens to characterize the enteric nervous system injury that occurs in necrotizing enterocolitis, and then used an animal model of experimental necrotizing enterocolitis to determine whether transplantation of neural stem cells can protect the enteric nervous system from injury.

Methods: Human intestinal specimens resected from patients with necrotizing enterocolitis (n = 18), from control patients with bowel atresia (n = 8), and from necrotizing enterocolitis and control patients undergoing stoma closure several months later (n = 14 and n = 6 respectively) were subjected to histologic examination, immunohistochemistry, and real-time reverse-transcription polymerase chain reaction to examine the myenteric plexus structure and neurotransmitter expression. In addition, experimental necrotizing enterocolitis was induced in newborn rat pups and neurotransplantation was performed by administration of fluorescently labeled neural stem cells, with subsequent visualization of transplanted cells and determination of intestinal integrity and intestinal motility.

Results: There was significant enteric nervous system damage with increased enteric nervous system apoptosis, and decreased neuronal nitric oxide synthase expression in myenteric ganglia from human intestine resected for necrotizing enterocolitis compared with control intestine. Structural and functional abnormalities persisted months later at the time of stoma closure. Similar abnormalities were identified in rat pups exposed to experimental necrotizing enterocolitis. Pups receiving neural stem cell transplantation had improved enteric nervous system and intestinal integrity, differentiation of transplanted neural stem cells into functional neurons, significantly improved intestinal transit, and significantly decreased mortality compared with control pups.

Conclusions: Significant injury to the enteric nervous system occurs in both human and experimental necrotizing enterocolitis. Neural stem cell transplantation may represent a novel future therapy for patients with necrotizing enterocolitis.
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http://dx.doi.org/10.1186/scrt387DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4054965PMC
October 2014

Synergistic effects of HB-EGF and mesenchymal stem cells in a murine model of intestinal ischemia/reperfusion injury.

J Pediatr Surg 2013 Jun;48(6):1323-9

The Research Institute at Nationwide Children's Hospital, Center for Perinatal Research, Columbus, OH 43205, USA.

Background: We have previously demonstrated that heparin-binding EGF-like growth factor (HB-EGF) administration protects the intestines from ischemia/reperfusion (I/R) injury in vivo. We have also shown that HB-EGF promotes mesenchymal stem cell (MSC) proliferation and migration in vitro. The goals of the current study were to examine the effects of HB-EGF and both bone marrow (BM)- and amniotic fluid (AF)-derived MSC on intestinal I/R injury in vivo.

Materials And Methods: MSC were isolated from pan-EGFP mice, expanded, and purified. Pluripotency was confirmed by induced differentiation. Mice were subjected to terminal ileum I/R and received either: (1) no therapy; (2) HB-EGF; (3) BM-MSC; (4) HB-EGF+BM-MSC; (5) AF-MSC; or (6) HB-EGF+AF-MSC. MSC engraftment, histologic injury, and intestinal permeability were quantified.

Results: There was increased MSC engraftment into injured compared to uninjured intestine for all experimental groups, with significantly increased engraftment for AF-MSC+HB-EGF compared to AF-MSC alone. Administration of HB-EGF and MSC improved intestinal histology and intestinal permeability after I/R injury. The greatest improvement was with combined administration of HB-EGF+AF-MSC.

Conclusions: Both HB-EGF alone and MSC alone can protect the intestines from I/R injury, with synergistic efficacy occurring when HB-EGF and AF-MSC are administered together.
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http://dx.doi.org/10.1016/j.jpedsurg.2013.03.032DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3710437PMC
June 2013

Heparin-binding EGF-like growth factor (HB-EGF) protects the intestines from radiation therapy-induced intestinal injury.

J Pediatr Surg 2013 Jun;48(6):1316-22

The Center for Perinatal Research, The Research Institute at Nationwide Children's Hospital, The Department of Pediatric Surgery, Nationwide Children's Hospital, Columbus, OH 43205, USA.

Purpose: Radiation therapy (RT) often induces enteritis by inhibiting proliferation and inducing apoptosis. Heparin-binding EGF-like growth factor (HB-EGF) has been shown to protect the intestine in several animal injury models. The objective of this study was to examine whether HB-EGF affects RT-induced intestinal injury.

Methods: HB-EGF or PBS was administered intraperitoneally to mice daily for 3 days, followed by total body irradiation (TBI). Three days after TBI, intestinal segments were harvested, and BrdU immunohistochemistry was performed to identify proliferating crypts (n=25). Four days after TBI, intestinal segments were harvested and assessed for histologic injury (n=34), and FITC-dextran was administered via gavage with serum FITC-dextran levels quantified to determine gut barrier function (n=18).

Results: Compared to non-HB-EGF-treated irradiated mice, administration of HB-EGF to irradiated mice led to a significantly increased percentage of proliferative crypts (72.6% vs. 50.5%, p=0.001), a significantly decreased percent of histologic sections with severe histologic injury (13.7% vs. 20.3%, p=0.005), and significantly reduced intestinal permeability (18.8 μg/mL vs. 22.6 μg/mL, p=0.02).

Conclusions: These results suggest that administration of HB-EGF protects the intestines from injury after exposure to radiation therapy. Administration of HB-EGF may represent a novel therapy for the prevention of radiation enteritis in the future.
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http://dx.doi.org/10.1016/j.jpedsurg.2013.03.030DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3710435PMC
June 2013

Heparin-binding epidermal growth factor-like growth factor attenuates acute lung injury and multiorgan dysfunction after scald burn.

J Surg Res 2013 Nov 12;185(1):329-37. Epub 2013 Jun 12.

Division of Pediatric Critical Care Medicine, Nationwide Children's Hospital, Columbus, Ohio; Center for Perinatal Research, The Research Institute at Nationwide Children's Hospital, Columbus, Ohio. Electronic address:

Background: Impaired gut barrier function and acute lung injury (ALI) are significant components of the multiorgan dysfunction syndrome that accompanies severe burns. Heparin-binding epidermal growth factor-like growth factor (HB-EGF) has been shown to reduce inflammation, preserve gut barrier function, and protect the lungs from acute injury in several models of intestinal injury; however, comparable effects of HB-EGF after burn injury have never been investigated. The present studies were based on the hypothesis that HB-EGF would reduce the severity of ALI and multiorgan dysfunction after scald burns in mice.

Materials And Methods: Mice were randomized to sham, burn (25% of total body surface area with full thickness dorsal scald), and burn + HB-EGF groups. The HB-EGF group was pretreated with two enteral doses of HB-EGF (1200 μg/kg/dose). Mice were resuscitated after injury and sacrificed at 8 h later. Their lungs were harvested for determination of pulmonary myeloperoxidase activity, wet:dry ratios, and terminal deoxynucleotidyl transferase dUTP nick end label and cleaved caspase 3 immunohistochemistry. Lung function was assessed using the SCIREQ Flexivent. Splenic apoptosis was quantified by Western blot for cleaved caspase 3, and intestinal permeability was measured using the everted gut sac method.

Results: Mice subjected to scald burn injury had increased lung myeloperoxidase levels, increased pulmonary and splenic apoptosis, elevated airway resistance and bronchial reactivity, and increased intestinal permeability compared with sham mice. These abnormalities were significantly attenuated in mice that were subjected to scald burn injury but treated with enteral HB-EGF.

Conclusions: These data suggest that HB-EGF protects mice from ALI after scald burn and attenuates the severity of postburn multiorgan dysfunction.
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http://dx.doi.org/10.1016/j.jss.2013.05.064DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3805668PMC
November 2013

The role of the intestinal microcirculation in necrotizing enterocolitis.

Semin Pediatr Surg 2013 May;22(2):83-7

Department of Pediatric Surgery, ED321 Nationwide Children's Hospital, The Ohio State University College of Medicine, 700 Children's Dr, Columbus, Ohio 43205, USA.

Necrotizing enterocolitis (NEC) continues to be a devastating inflammatory disease of the newborn intestine. Despite advances in management, morbidity and mortality remain high. While it is clear that intestinal ischemia plays a large role in disease pathogenesis, attempts to link NEC to intestinal macrovascular derangement have been largely unsuccessful. More recently, there has been a concerted effort to characterize the pathologic changes of the intestinal microcirculation in response to intestinal injury, including NEC. This microcirculatory regulation is controlled by a balance of vasoconstrictor and vasodilator forces. Vasoconstriction is mediated primarily by endothelin-1 (ET-1), while vasodilation is mediated primarily by nitric oxide (NO). These chemical mediators have been implicated in many aspects of intestinal ischemic injury and NEC, with the balance shifting toward increased vasoconstriction associated with intestinal injury. With a proper understanding of these antagonistic forces, potential therapeutic avenues may result from improving this pathologic microcirculatory dysregulation.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3646103PMC
http://dx.doi.org/10.1053/j.sempedsurg.2013.01.004DOI Listing
May 2013

A technique for systemic mesenchymal stem cell transplantation in newborn rat pups.

J Invest Surg 2012 Dec;25(6):405-14

Department of Pediatric Surgery, College of Medicine, The Research Institute at Nationwide Children's Hospital, Center for Perinatal Research, Nationwide Children's Hospital, The Ohio State University, Columbus, OH 43205, USA.

Mesenchymal stem cells (MSC) have the potential to aid tissue regeneration. Intravenous (IV) MSC administration is currently being assessed following tissue injury. However, few studies have been performed to establish a safe and effective method of IV MSC infusion for newborns. We have established a safe, nontraumatic and effective technique for systemic MSC transplantation in newborn rats. Yellow-fluorescent-protein (YFP)-labeled MSC were characterized using MSC markers and their differentiation potential was confirmed. Rat pups were delivered by C-section on gestational day 21. The umbilical vein (UV) was cannulated and used for IV injection of MSC or saline control, which was performed under ultrasonographic imaging. An additional control group consisted of UV MSC injection in adult mice. Mean operating time, success rate of cannulation and death rate were recorded. YFP-MSC quantification in multiple organs was performed. Mean operating time was 3.9 ± 1.1 min. The success of UV MSC injection was 92.8%. The immediate and 24 hr delayed death rate for rat pups was significantly lower than that of adult mice (p < .05). No pups receiving saline injection died. After locating the patent foramen ovale (PFO) of newborn pups by ultrasonographic imaging, extra pulse-waves and wave-shape changes were detected when MSC were injected. The number of YFP-MSC was 15.8 ± 4.1 cells per visual field (CPVF) in the lungs, 2.9 ± 1.2 CPVF in the heart, and 19.8 ± 5.0 CPVF in the intestines. We conclude that IV MSC infusion through the UV is a convenient, safe, and effective method for systemic MSC transplantation in prematurely delivered newborn rats.
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http://dx.doi.org/10.3109/08941939.2012.661519DOI Listing
December 2012

Constructing manmade enzymes for oxygen activation.

Dalton Trans 2013 Mar 18;42(9):3136-50. Epub 2012 Oct 18.

School of Biochemistry, University of Bristol, University Walk, Bristol, BS8 1TD, UK.

Natural oxygenases catalyse the insertion of oxygen into an impressive array of organic substrates with exquisite efficiency, specificity and power unparalleled by current biomimetic catalysts. However, their true potential to provide tailor-made oxygenation catalysts remains largely untapped, perhaps a consequence of the evolutionary complexity imprinted into their three-dimensional structures through millennia of exposure to parallel selective pressures. In this perspective we describe how we may take inspiration from natural enzymes to design manmade oxygenase enzymes free from such complexity. We explore the differing chemistries accessed by natural oxygenases and outline a stepwise methodology whereby functional elements key to oxygenase catalysis are assembled within artificially designed protein scaffolds.
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http://dx.doi.org/10.1039/c2dt32010jDOI Listing
March 2013

Heparin-binding epidermal growth factor-like growth factor and mesenchymal stem cells act synergistically to prevent experimental necrotizing enterocolitis.

J Am Coll Surg 2012 Oct 21;215(4):534-45. Epub 2012 Jul 21.

The Research Institute at Nationwide Children's Hospital, Center for Perinatal Research, Department of Pediatric Surgery, Nationwide Children's Hospital, and the Ohio State University College of Medicine, Columbus, OH, USA.

Background: We have shown that administration of heparin-binding EGF (epidermal growth factor)-like growth factor (HB-EGF) protects the intestines from experimental necrotizing enterocolitis (NEC). We have also demonstrated that systemically administered mesenchymal stem cells (MSC) can engraft into injured intestines. This study investigated the effects of HB-EGF on MSC in vitro, and whether MSC and HB-EGF can act synergistically to prevent NEC in vivo.

Study Design: In vitro, the effect of HB-EGF on MSC proliferation, migration, and apoptosis was determined. In vivo, rat pups received MSC either intraperitoneally (IP) or intravenously (IV). Pups were assigned to 1 of 7 groups: Group 1, breast-fed; Group 2, experimental NEC; Group 3, NEC+HB-EGF; Group 4, NEC+MSC IP; Group 5, NEC+HB-EGF+MSC IP; Group 6, NEC+MSC IV; or Group 7, NEC+HB-EGF+MSC IV. Mesechymal stem cell engraftment, histologic injury, intestinal permeability, and mortality were determined.

Results: Heparin-binding EGF-like growth factor promoted MSC proliferation and migration, and decreased MSC apoptosis in vitro. In vivo, MSC administered IV had increased engraftment into NEC-injured intestine compared with MSC administered IP (p < 0.05). Heparin binding EGF-like growth factor increased engraftment of IP-administered MSC (p < 0.01) and IV-administered MSC (p < 0.05). Pups in Groups 3 to 7 had a decreased incidence of NEC compared with nontreated pups (Group 2), with the lowest incidence in pups treated with HB-EGF+MSC IV (p < 0.01). Pups in Group 7 had a significantly decreased incidence of intestinal dilation and perforation, and had the lowest intestinal permeability, compared with other treatment groups (p < 0.01). Pups in all experimental groups had significantly improved survival compared with pups exposed to NEC, with the best survival in Group 7 (p < 0.05).

Conclusions: Heparin-binding EGF-like growth factor and MSC act synergistically to reduce injury and improve survival in experimental NEC.
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http://dx.doi.org/10.1016/j.jamcollsurg.2012.05.037DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3444529PMC
October 2012

Heparin-binding epidermal growth factor-like growth factor protects mesenchymal stem cells.

J Surg Res 2012 Oct 23;177(2):359-64. Epub 2012 May 23.

Department of Pediatric Surgery, Research Institute at Nationwide Children's Hospital, Nationwide Children's Hospital, Center for Perinatal Research, Ohio State University College of Medicine, Columbus, Ohio, USA.

Background: We have previously demonstrated that mesenchymal stem cell (MSC) administration protects the intestines from injury in a mouse model of intestinal ischemia/reperfusion injury. We have also shown that heparin-binding epidermal growth factor-like growth factor (HB-EGF) is a potent intestinal cytoprotective agent in vivo that can protect the intestines by way of its effects on stem cells. The goal of the present study was to examine the effects of HB-EGF on both amniotic fluid (AF)- and bone marrow (BM)-derived MSCs in vitro.

Methods: MSCs were isolated from the AF and BM of pan-EGFP mice, grown in MSC-specific culture medium, and purified by sequential passages according to their adherence properties. Pluripotency was confirmed by induced differentiation. After incubation of MSCs with HB-EGF, proliferation was quantified using the CyQuant cell proliferation assay kit under normoxic and anoxic conditions. Chemotaxis was quantified using the CHEMICON QCM cell migration kit, and apoptosis was determined using caspase-3 immunohistochemistry after exposure of the MSCs to anoxic stress.

Results: AF-MSCs and BM-MSCs showed significantly increased proliferation and migration in response to HB-EGF. HB-EGF significantly protected AF-MSCs and BM-MSCs from anoxia-induced apoptosis. The proliferative and anti-apoptotic effects of HB-EGF were even more pronounced in AF-MSCs than in BM-MSCs.

Conclusions: These results have demonstrated that HB-EGF acts as a mitogenic and chemotactic agent for MSCs that protects MSCs from injury. These findings could have important implications for future experiments designed to use MSCs to protect the intestines from injury.
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http://dx.doi.org/10.1016/j.jss.2012.05.016DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3434250PMC
October 2012

Near infrared laser-tissue welding using nanoshells as an exogenous absorber.

Lasers Surg Med 2005 Aug;37(2):123-9

Department of Bioengineering, Rice University, 6100 S. Main Street, Houston, TX 77251, USA.

Background And Objective: Gold nanoshells are a new class of nanoparticles that can be designed to strongly absorb light in the near infrared (NIR). These particles provide much larger absorption cross-sections and efficiency than can be achieved with currently used chemical chromophores without photobleaching. In these studies, we have investigated the use of gold nanoshells as exogenous NIR absorbers to facilitate NIR laser-tissue welding.

Study Design/materials And Methods: Gold nanoshells with peak extinction matching the NIR wavelength of the laser being used were manufactured and suspended in an albumin solder. Optimization work was performed on ex vivo muscle samples and then translated into testing in an in vivo rat skin wound-healing model. Mechanical testing of the muscle samples was immediately performed and compared to intact tissue mechanical properties. In the in vivo study, full thickness incisions in the dorsal skin of rats were welded, and samples of skin were excised at 0, 5, 10, 21, and 32 days for analysis of strength and wound healing response.

Results: Mechanical testing of nanoshell-solder welds in muscle revealed successful fusion of tissues with tensile strengths of the weld site equal to the uncut tissue. No welding was accomplished with this light source when using solder formulations without nanoshells. Mechanical testing of the skin wounds showed sufficient strength for closure and strength increased over time. Histological examination showed good wound-healing response in the soldered skin.

Conclusions: The use of nanoshells as an exogenous absorber allows the usage of light sources that are minimally absorbed by tissue components, thereby, minimizing damage to surrounding tissue and allowing welding of thicker tissues.
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http://dx.doi.org/10.1002/lsm.20206DOI Listing
August 2005

Evaluation of misting controls to reduce respirable silica exposure for brick cutting.

Ann Occup Hyg 2005 Aug 21;49(6):503-10. Epub 2005 Apr 21.

University of Wisconsin-Stout, PO Box 790, Menomonie, WI 54751, USA.

It is estimated that more than 1.7 million workers in the United States are potentially exposed to respirable crystalline silica, with a large percentage having been exposed to silica concentrations higher than the limits set by current standards and regulations. The purpose of this study is to characterize the use of water-misting engineering controls to reduce exposure to respirable crystalline silica for construction workers engaged in the task of brick cutting. Since data concerning the efficacy of engineering controls collected at worksites is often confounded by factors such as wind, worker skill level, the experiments were conducted in a laboratory environment. A completely enclosed testing chamber housed the brick-cutting saw. Respirable dust concentrations were measured using the Model 3321 Aerodynamic Particle Sizer. Specifically, the laboratory experiment was designed to compare dust suppression through water misting using conventional freely flowing water techniques. Brass atomizing nozzles with three flow rates were used for making this comparison: low (5.0 ml s(-1) or 4.8 gal h(-1)), medium (9.0 ml s(-1) or 8.6 gal h(-1)) and high (18 ml s(-1) or 17.3 gal h(-1)). The flow rate for freely flowing water, using manufacturer-supplied equipment, was 50 ml s(-1) (48 gal h(-1)). The experiment consisted of five replications of five samples each (low-misting, medium-misting, high-misting, freely flowing water and no control). The order of sampling within each replicate was randomized. Estimates of dust reduction showed that low-misting nozzles reduced the respirable mass fraction of dust by about 63%, medium-misting nozzles by about 67%, high-misting nozzles by about 79% and freely flowing water by about 93%. Based on these results, it may be feasible to use misting to control respirable silica dust instead of freely flowing water. This strategy is of practical interest to the construction industry which must frequently limit the amount of water used on construction sites.
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http://dx.doi.org/10.1093/annhyg/mei011DOI Listing
August 2005
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