@article{leavens_tell_kissell_smith_smith_wagner_shelver_wu_baynes_riviere_et al._2014, title={Development of a physiologically based pharmacokinetic model for flunixin in cattle (Bos taurus)}, volume={31}, ISSN={["1944-0057"]}, DOI={10.1080/19440049.2014.938363}, abstractNote={Frequent violation of flunixin residues in tissues from cattle has been attributed to non-compliance with the USFDA-approved route of administration and withdrawal time. However, the effect of administration route and physiological differences among animals on tissue depletion has not been determined. The objective of this work was to develop a physiologically based pharmacokinetic (PBPK) model to predict plasma, liver and milk concentrations of flunixin in cattle following intravenous (i.v.), intramuscular (i.m.) or subcutaneous (s.c.) administration for use as a tool to determine factors that may affect the withdrawal time. The PBPK model included blood flow-limited distribution in all tissues and elimination in the liver, kidney and milk. Regeneration of parent flunixin due to enterohepatic recirculation and hydrolysis of conjugated metabolites was incorporated in the liver compartment. Values for physiological parameters were obtained from the literature, and partition coefficients for all tissues but liver and kidney were derived empirically. Liver and kidney partition coefficients and elimination parameters were estimated for 14 pharmacokinetic studies (including five crossover studies) from the literature or government sources in which flunixin was administered i.v., i.m. or s.c. Model simulations compared well with data for the matrices following all routes of administration. Influential model parameters included those that may be age or disease-dependent, such as clearance and rate of milk production. Based on the model, route of administration would not affect the estimated days to reach the tolerance concentration (0.125 mg kg−1) in the liver of treated cattle. The majority of USDA-reported violative residues in liver were below the upper uncertainty predictions based on estimated parameters, which suggests the need to consider variability due to disease and age in establishing withdrawal intervals for drugs used in food animals. The model predicted that extravascular routes of administration prolonged flunixin concentrations in milk, which could result in violative milk residues in treated cattle.}, number={9}, journal={FOOD ADDITIVES AND CONTAMINANTS PART A-CHEMISTRY ANALYSIS CONTROL EXPOSURE & RISK ASSESSMENT}, author={Leavens, Teresa and Tell, L. A. and Kissell, L. W. and Smith, G. W. and Smith, D. J. and Wagner, S. A. and Shelver, W. L. and Wu, H. L. and Baynes, R. E. and Riviere, J. E. and et al.}, year={2014}, month={Sep}, pages={1506–1521} }
 @article{wu_baynes_leavens_tell_riviere_2013, title={Use of population pharmacokinetic modeling and Monte Carlo simulation to capture individual animal variability in the prediction of flunixin withdrawal times in cattle}, volume={36}, ISSN={0140-7783}, url={http://dx.doi.org/10.1111/j.1365-2885.2012.01420.x}, DOI={10.1111/j.1365-2885.2012.01420.x}, abstractNote={The objective of this study was to develop a population pharmacokinetic (PK) model and predict tissue residues and the withdrawal interval (WDI) of flunixin in cattle. Data were pooled from published PK studies in which flunixin was administered through various dosage regimens to diverse populations of cattle. A set of liver data used to establish the regulatory label withdrawal time (WDT) also were used in this study. Compartmental models with first‐order absorption and elimination were fitted to plasma and liver concentrations by a population PK modeling approach. Monte Carlo simulations were performed with the population mean and variabilities of PK parameters to predict liver concentrations of flunixin. The PK of flunixin was described best by a 3‐compartment model with an extra liver compartment. The WDI estimated in this study with liver data only was the same as the label WDT. However, a longer WDI was estimated when both plasma and liver data were included in the population PK model. This study questions the use of small groups of healthy animals to determine WDTs for drugs intended for administration to large diverse populations. This may warrant a reevaluation of the current procedure for establishing WDT to prevent violative residues of flunixin.}, number={3}, journal={Journal of Veterinary Pharmacology and Therapeutics}, publisher={Wiley}, author={Wu, H. and Baynes, R. E. and Leavens, T. and Tell, L. A. and Riviere, J. E.}, year={2013}, month={Jun}, pages={248–257} }
 @article{riviere_leavens_brooks_monteiro-riviere_2012, title={Acute vascular effects of nanoparticle infusion in isolated perfused skin}, volume={8}, ISSN={1549-9634}, url={http://dx.doi.org/10.1016/j.nano.2012.02.016}, DOI={10.1016/j.nano.2012.02.016}, abstractNote={The majority of studies on the effect of nanomaterials on biological function involves either isolated in vitro cell systems or are concerned with in vivo effects after inhalational or dermal exposure. The current work reports on an intriguing observation of the vascular effects seen in an ex vivo perfused tissue preparation, the isolated perfused porcine skin flap (IPPSF), in studies conducted to assess nanomaterial biodistribution. Compared with a relatively large dataset involving organic chemical infusions (n = 53), infusion of six different nanoparticles of diverse sizes and composition (silica or dextran coated Fe(2)O(3), silica or citrate coated silver, PEG or carboxylated quantum dots [QD]) resulted in statistically significant post-infusion flap weight gain and an increase in arterial perfusion pressure (especially with QD-PEG). In contrast, infusion with nC(60) nanoparticles did not produce these effects. These observations suggest certain nanoparticle infusions may be associated with acute vascular physiologic effects that merit further attention.In this study utilizing a perfused porcine skin flap, specific nanoparticle infusions were demonstrated to be associated with significant acute vascular physiological effects.}, number={4}, journal={Nanomedicine: Nanotechnology, Biology and Medicine}, publisher={Elsevier BV}, author={Riviere, Jim E. and Leavens, Teresa L. and Brooks, James D. and Monteiro-Riviere, Nancy A.}, year={2012}, month={May}, pages={428–431} }
 @article{leavens_tell_clothier_griffith_baynes_riviere_2012, title={Development of a physiologically based pharmacokinetic model to predict tulathromycin distribution in goats}, volume={35}, ISSN={["1365-2885"]}, DOI={10.1111/j.1365-2885.2011.01304.x}, abstractNote={Leavens, T. L., Tell, L. A., Clothier, K. A., Griffith, R. W., Baynes, R. E., Riviere, J. E. Development of a physiologically based pharmacokinetic model to predict tulathromycin distribution in goats. J. vet. Pharmacol. Therap. 35, 121–131.}, number={2}, journal={JOURNAL OF VETERINARY PHARMACOLOGY AND THERAPEUTICS}, author={Leavens, T. L. and Tell, L. A. and Clothier, K. A. and Griffith, R. W. and Baynes, R. E. and Riviere, J. E.}, year={2012}, month={Apr}, pages={121–131} }
 @article{leavens_monteiro-riviere_inman_brooks_oldenburg_riviere_2012, title={In vitro biodistribution of silver nanoparticles in isolated perfused porcine skin flaps}, volume={32}, ISSN={["0260-437X"]}, url={http://www.scopus.com/inward/record.url?eid=2-s2.0-84866866954&partnerID=MN8TOARS}, DOI={10.1002/jat.2750}, abstractNote={ABSTRACT}, number={11}, journal={JOURNAL OF APPLIED TOXICOLOGY}, author={Leavens, Teresa L. and Monteiro-Riviere, Nancy A. and Inman, Alfred O. and Brooks, James D. and Oldenburg, Steven J. and Riviere, Jim E.}, year={2012}, month={Nov}, pages={913–919} }
 @article{romanet_smith_leavens_baynes_wetzlich_riviere_tell_2012, title={Pharmacokinetics and tissue elimination of tulathromycin following subcutaneous administration in meat goats}, volume={73}, ISSN={["0002-9645"]}, DOI={10.2460/ajvr.73.10.1634}, abstractNote={Abstract}, number={10}, journal={AMERICAN JOURNAL OF VETERINARY RESEARCH}, author={Romanet, Jessica and Smith, Geof W. and Leavens, Teresa L. and Baynes, Ronald E. and Wetzlich, Scott E. and Riviere, Jim E. and Tell, Lisa A.}, year={2012}, month={Oct}, pages={1634–1640} }
 @article{barchowsky_buckley_carlson_fitsanakis_ford_genter_germolec_leavens_lehman-mckeeman_safe_et al._2012, title={The Toxicology Education Summit: Building the Future of Toxicology Through Education}, volume={127}, ISSN={["1096-6080"]}, DOI={10.1093/toxsci/kfs111}, abstractNote={Toxicology and careers in toxicology, as well as many other scientific disciplines, are undergoing rapid and dramatic changes as new discoveries, technologies, and hazards advance at a blinding rate. There are new and ever increasing demands on toxicologists to keep pace with expanding global economies, highly fluid policy debates, and increasingly complex global threats to public health. These demands must be met with new paradigms for multidisciplinary, technologically complex, and collaborative approaches that require advanced and continuing education in toxicology and associated disciplines. This requires paradigm shifts in educational programs that support recruitment, development, and training of the modern toxicologist, as well as continued education and retraining of the midcareer professional to keep pace and sustain careers in industry, government, and academia. The Society of Toxicology convened the Toxicology Educational Summit to discuss the state of toxicology education and to strategically address educational needs and the sustained advancement of toxicology as a profession. The Summit focused on core issues of: building for the future of toxicology through educational programs; defining education and training needs; developing the "Total Toxicologist"; continued training and retraining toxicologists to sustain their careers; and, finally, supporting toxicology education and professional development. This report summarizes the outcomes of the Summit, presents examples of successful programs that advance toxicology education, and concludes with strategies that will insure the future of toxicology through advanced educational initiatives.}, number={2}, journal={TOXICOLOGICAL SCIENCES}, author={Barchowsky, Aaron and Buckley, Lorrene A. and Carlson, Gary P. and Fitsanakis, Vanessa A. and Ford, Sue M. and Genter, Mary Beth and Germolec, Dori R. and Leavens, Teresa L. and Lehman-McKeeman, Lois D. and Safe, Stephen H. and et al.}, year={2012}, month={Jun}, pages={331–338} }
 @article{clothier_leavens_griffith_wetzlich_baynes_riviere_tell_2012, title={Tulathromycin assay validation and tissue residues after single and multiple subcutaneous injections in domestic goats (Capra aegagrus hircus)}, volume={35}, ISSN={["1365-2885"]}, DOI={10.1111/j.1365-2885.2011.01300.x}, abstractNote={Clothier, K. A., Leavens, T., Griffith, R. W., Wetzlich, S. E., Baynes, R. E., Riviere, J. E., Tell, L. A. Tulathromycin assay validation and tissue residues after single and multiple subcutaneous injections in domestic goats (Capra aegagrus hircus). J. vet. Pharmacol. Therap. 35, 113–120.}, number={2}, journal={JOURNAL OF VETERINARY PHARMACOLOGY AND THERAPEUTICS}, author={Clothier, K. A. and Leavens, T. and Griffith, R. W. and Wetzlich, S. E. and Baynes, R. E. and Riviere, J. E. and Tell, L. A.}, year={2012}, month={Apr}, pages={113–120} }
 @article{clothier_leavens_griffith_wetzlich_baynes_riviere_tell_2011, title={Pharmacokinetics of tulathromycin after single and multiple subcutaneous injections in domestic goats (Capra aegagrus hircus)}, volume={34}, ISSN={["0140-7783"]}, DOI={10.1111/j.1365-2885.2010.01261.x}, abstractNote={Clothier, K. A., Leavens, T., Griffith, R. W., Wetzlich, S. E., Baynes, R. E., Riviere, J. E., Tell, L. A. Pharmacokinetics of tulathromycin after single and multiple subcutaneous injections in domestic goats (Capra aegagrus hircus). J. vet. Pharmacol. Therap.34, 448–454.}, number={5}, journal={JOURNAL OF VETERINARY PHARMACOLOGY AND THERAPEUTICS}, author={Clothier, K. A. and Leavens, T. and Griffith, R. W. and Wetzlich, S. E. and Baynes, R. E. and Riviere, J. E. and Tell, L. A.}, year={2011}, month={Oct}, pages={448–454} }
 @article{young_smith_leavens_wetzlich_baynes_mason_riviere_tell_2011, title={Pharmacokinetics of tulathromycin following subcutaneous administration in meat goats}, volume={90}, ISSN={["0034-5288"]}, DOI={10.1016/j.rvsc.2010.06.025}, abstractNote={Tulathromycin is a triamilide antibiotic that maintains therapeutic concentrations for an extended period of time. The drug is approved for the treatment of respiratory disease in cattle and swine and is occasionally used in goats. To investigate the pharmacokinetics of tulathromycin in meat goats, 10 healthy Boer goats were administered a single 2.5 mg/kg subcutaneous dose of tulathromycin. Plasma concentrations were measured by ultra-high pressure liquid chromatography tandem mass spectrometry (UPLC–MS/MS) detection. Plasma maximal drug concentration (Cmax) was 633 ± 300 ng/ml (0.40 ± 0.26 h post-subcutaneous injection). The half-life of tulathromycin in goats was 110 ± 19.9 h. Tulathromycin was rapidly absorbed and distributed widely after subcutaneous injection 33 ± 6 L/kg. The mean AUC of the group was 12,500 ± 2020 h ng/mL for plasma. In this study, it was determined that the pharmacokinetics of tulathromycin after a single 2.5 mg/kg SC injection in goats were very similar to what has been previously reported in cattle.}, number={3}, journal={RESEARCH IN VETERINARY SCIENCE}, author={Young, Gabrielle and Smith, Geof W. and Leavens, Teresa L. and Wetzlich, Scott E. and Baynes, Ronald E. and Mason, Sharon E. and Riviere, Jim E. and Tell, Lisa A.}, year={2011}, month={Jun}, pages={477–479} }
 @article{leavens_xia_lee_monteiro-riviere_brooks_riviere_2010, title={Evaluation of perfused porcine skin as a model system to quantitate tissue distribution of fullerene nanoparticles}, volume={197}, ISSN={["1879-3169"]}, url={http://gateway.webofknowledge.com/gateway/Gateway.cgi?GWVersion=2&SrcAuth=ORCID&SrcApp=OrcidOrg&DestLinkType=FullRecord&DestApp=WOS_CPL&KeyUT=WOS:000279989500001&KeyUID=WOS:000279989500001}, DOI={10.1016/j.toxlet.2010.03.1119}, abstractNote={Nanomaterials are increasingly playing a role in society for uses ranging from biomedicine to microelectronics, however pharmacokinetic studies, which will be necessary for human health risk assessments, are limited. Tissue distribution, one component of pharmacokinetics, can be assessed by quantifying arterial extraction of materials in an isolated perfused porcine skin flap (IPPSF). The objective of this study was to assess the IPPSF as a model system to quantitate the distribution of fullerene nanoparticles (nC60) from the vascular space into tissues. IPPSFs were perfused for 4 h with 0.885 μg/mL nC60 in media with immunoglobulin G present (IgG+) or absent (IgG−) followed by a 4 h perfusion with media only during a washout phase. Arterial and venous concentrations of nC60 were measured in the media by HPLC–UV/vis chromatography. Steady state differences in the arterial and venous nC60 concentrations were compared to determine extraction from the vascular space of the IPPSF, and the venous nC60 concentration versus time profiles were used to calculate compartmental pharmacokinetic parameters. The steady state differences in the arterial and venous concentrations in the IPPSF were small with extraction percentages (mean ± sd) of 8.2 ± 5.7% and 4.2 ± 6.7% for IgG+ and IgG− media, respectively, and were not significantly different between the types of media. The venous concentrations of nC60 in both types of media were best fit with a 2 compartment model with terminal half lives (harmonic mean) of 17.5 and 28.0 min for IgG+ and IgG− media, respectively. The apparent volumes of distribution at steady state were 0.12 ± 0.047 and 0.10 ± 0.034 L/kg, for IgG+ and IgG− media, respectively. By 4 h following infusion of nC60, the recovery of nC60 in the venous effluent was 94 ± 5.5% and 97 ± 6.8% of the infused nC60 for IgG+ and IgG− media, respectively. Based on the apparent volume of distribution, the low extraction during the perfusion, and the high percentage recovery following the washout phase, there was limited distribution of nC60 from the vascular space into the extracellular space and negligible intracellular uptake of nC60 in this system.}, number={1}, journal={TOXICOLOGY LETTERS}, author={Leavens, Teresa L. and Xia, Xin Rui and Lee, Hyun A. and Monteiro-Riviere, Nancy A. and Brooks, James D. and Riviere, Jim E.}, year={2010}, month={Aug}, pages={1–6} }
 @article{radcliffe_leavens_wagner_olabisi_struve_wong_tewksbury_chapman_dorman_2010, title={Pharmacokinetics of radiolabeled tungsten (W-188) in male Sprague-Dawley rats following acute sodium tungstate inhalation}, volume={22}, ISSN={["0895-8378"]}, DOI={10.3109/08958370902913237}, abstractNote={Aerosol cloud formation may occur when certain tungsten munitions strike hard targets, placing military personnel at increased risk of exposure. Although the pharmacokinetics of various forms of tungsten have been studied in animals following intravenous and oral administration, tungsten disposition following inhalation remains incompletely characterized. The objective of this study was to evaluate the pharmacokinetics of inhaled tungstate (WO4) in rats. Male, 16-wk-old, CD rats (n = 7 rats/time point) underwent a single, 90-min, nose-only exposure to an aerosol (mass median aerodynamic diameter [MMAD] 1.50 μm ) containing 256 mg W/m3 as radiolabeled sodium tungstate (Na2188WO4). 188W tissue concentrations were determined at 0, 1, 3, 7, and 21 days postexposure by gamma spectrometry. The thyroid and urine had the highest 188W levels postexposure, and urinary excretion was the primary route of 188W elimination. The pharmacokinetics of tungsten in most tissues was best described with a two-compartment pharmacokinetic model with initial phase half-lives of approximately 4 to 6 h and a longer terminal phase with half-lives of approximately 6 to 67 days. The kidney, adrenal, spleen, femur, lymph nodes, and brain continued to accumulate small amounts of tungsten as reflected by tissue:blood activity ratios that increased throughout the 21-day period. At day 21 all tissues except the thyroid, urine, lung, femur, and spleen had only trace levels of 188W. Data from this study can be used for development and refinement of pharmacokinetic models for tungsten inhalation exposure in environmental and occupational settings.}, number={1}, journal={INHALATION TOXICOLOGY}, author={Radcliffe, Pheona M. and Leavens, Teresa L. and Wagner, Dean J. and Olabisi, Ayodele O. and Struve, Melanie F. and Wong, Brian A. and Tewksbury, Earl and Chapman, Gail D. and Dorman, David C.}, year={2010}, month={Jan}, pages={69–76} }
 @article{borghoff_parkinson_leavens_2010, title={Physiologically based pharmacokinetic rat model for methyl tertiary-butyl ether; comparison of selected dose metrics following various MTBE exposure scenarios used for toxicity and carcinogenicity evaluation}, volume={275}, ISSN={["0300-483X"]}, DOI={10.1016/j.tox.2010.06.003}, abstractNote={There are a number of cancer and toxicity studies that have been carried out to assess hazard from methyl tertiary-butyl ether (MTBE) exposure via inhalation and oral administration. MTBE has been detected in surface as well as ground water supplies which emphasized the need to assess the risk from exposure via drinking water contamination. This model can now be used to evaluate route-to-route extrapolation issues concerning MTBE exposures but also as a means of comparing potential dose metrics that may provide insight to differences in biological responses observed in rats following different routes of MTBE exposure. Recently an updated rat physiologically based pharmacokinetic (PBPK) model was published that relied on a description of MTBE and its metabolite tertiary-butyl alcohol (TBA) binding to alpha 2u-globulin, a male rat-specific protein. This model was used to predict concentrations of MTBE and TBA in the kidney, a target tissue in the male rat. The objective of this study was to use this model to evaluate the dosimetry of MTBE and TBA in rats following different exposure scenarios, used to evaluate the toxicity and carcinogenicity of MTBE, and compare various dose metrics under these different conditions. Model simulations suggested that although inhalation and drinking water exposures show a similar pattern of MTBE and TBA exposure in the blood and kidney (i.e. concentration-time profiles), the total blood and kidney levels following exposure of MTBE to 7.5mg/ml MTBE in the drinking water for 90 days is in the same range as administration of an oral dose of 1000 mg/kg MTBE. Evaluation of the dose metrics also supports that a high oral bolus dose (i.e. 1000 mg/kg MTBE) results in a greater percentage of the dose exhaled as MTBE with a lower percent metabolized to TBA as compared to dose of MTBE that is delivered over a longer period of time as in the case of drinking water.}, number={1-3}, journal={TOXICOLOGY}, author={Borghoff, Susan J. and Parkinson, Horace and Leavens, Teresa L.}, year={2010}, month={Sep}, pages={79–91} }
 @article{radcliffe_olabisi_wagner_leavens_wong_struve_chapman_wilfong_dorman_2009, title={Acute sodium tungstate inhalation is associated with minimal olfactory transport of tungsten (W-188) to the rat brain}, volume={30}, ISSN={["0161-813X"]}, DOI={10.1016/j.neuro.2009.02.004}, abstractNote={Olfactory transport of represents an important mechanism for direct delivery of certain metals to the central nervous system (CNS). The objective of this study was to determine whether inhaled tungsten (W) undergoes olfactory uptake and transport to the rat brain. Male, 16-week-old, Sprague–Dawley rats underwent a single, 90-min, nose-only exposure to a Na2188WO4 aerosol (256 mg W/m3). Rats had the right nostril plugged to prevent nasal deposition of 188W on the occluded side. The left and right sides of the nose and brain, including the olfactory pathway and striatum, were sampled at 0, 1, 3, 7, and 21 days post-exposure. Gamma spectrometry (n = 7 rats/time point) was used to compare the levels of 188W found on the left and right sides of the nose and brain and blood to determine the contribution of olfactory uptake to brain 188W levels. Respiratory and olfactory epithelial samples from the side with the occluded nostril had significantly lower end-of-exposure 188W levels confirming the occlusion procedure. Olfactory bulb, olfactory tract/tubercle, striatum, cerebellum, rest of brain 188W levels paralleled blood 188W concentrations at approximately 2–3% of measured blood levels. Brain 188W concentrations were highest immediately following exposure, and returned to near background concentrations within 3 days. A statistically significant difference in olfactory bulb 188W concentration was seen at 3 days post-exposure. At this time, 188W concentrations in the olfactory bulb from the side ipsilateral to the unoccluded nostril were approximately 4-fold higher than those seen in the contralateral olfactory bulb. Our data suggest that the concentration of 188W in the olfactory bulb remained low throughout the experiment, i.e., approximately 1–3% of the amount of tungsten seen in the olfactory epithelium suggesting that olfactory transport plays a minimal role in delivering tungsten to the rat brain.}, number={3}, journal={NEUROTOXICOLOGY}, author={Radcliffe, Pheona M. and Olabisi, Ayodele O. and Wagner, Dean J. and Leavens, Teresa and Wong, Brian A. and Struve, Melanie F. and Chapman, Gail D. and Wilfong, Erin R. and Dorman, David C.}, year={2009}, month={May}, pages={445–450} }
 @article{leavens_borghoff_2009, title={Physiologically Based Pharmacokinetic Model of Methyl Tertiary Butyl Ether and Tertiary Butyl Alcohol Dosimetry in Male Rats Based on Binding to alpha 2u-Globulin}, volume={109}, ISSN={["1096-0929"]}, DOI={10.1093/toxsci/kfp049}, abstractNote={Current physiologically based pharmacokinetic (PBPK) models for the fuel additive methyl tertiary butyl ether (MTBE) and its metabolite tertiary butyl alcohol (TBA) have not included a mechanism for chemical binding to the male rat-specific protein alpha2u-globulin, which has been postulated to be responsible for renal effects in male rats observed in toxicity and carcinogenicity studies with MTBE. The objective of this work was to expand the previously published models for MTBE to include binding to alpha2u-globulin in the kidney of male rats. In the model, metabolism of MTBE was assumed to occur only in the liver via two saturable pathways. TBA metabolism was assumed to occur only in the liver via one saturable, low-affinity pathway and to be inducible following repeated exposures. The binding of MTBE and TBA to alpha2u-globulin was modeled as saturable and competitive and was assumed to only affect the rate of hydrolysis of alpha2u-globulin in the kidney. The developed model characterized the differences in kidney concentrations of MTBE and TBA in male versus female rats from inhalation exposures to MTBE, as well as the observed changes in blood and tissue concentrations from repeated exposure to TBA. The model-predicted binding affinity of MTBE to alpha2u-globulin was greater than TBA, and the hydrolysis rate of chemically bound alpha2u-globulin was approximately 30% of the unbound protein. This PBPK model supports the role of MTBE and TBA binding to the male rat-specific protein alpha2u-globulin as essential for predicting concentrations of these chemicals in the kidney following exposure.}, number={2}, journal={TOXICOLOGICAL SCIENCES}, author={Leavens, Teresa L. and Borghoff, Susan J.}, year={2009}, month={Jun}, pages={321–335} }