Health Phys. Abstracts，Volume 121,Number 4

TheNaturalHistoryofAcuteRadiation-inducedH-ARSandConcomitantMulti-organInjuryintheNon-humanPrimate:TheMCARTExperience

Ann M. Farese1, Catherine Booth2, Greg L. Tudor2, Wanchang Cui3, Eric P. Cohen1, George A. Parker4, Kim G. Hankey1, Thomas J. MacVittie1

(1.University of Maryland, School of Medicine, Baltimore, MD

2.Epistem, LTD, Manchester, England;

3.Armed Forces Radiobiology Research Institute, Bethesda, MD;

4.Charles River Laboratories, Pathology Associates, Durham, NC)

Abstract:The dose response relationship and corresponding values for mid-lethal dose and slope are used to define the dose- and time-dependent parameters of the hematopoietic acute radiation syndrome. The characteristic time course of mortality, morbidity, and secondary endpoints are well defined. The concomitant comorbidities, potential mortality, and other multi-organ injuries that are similarly dose- and time-dependent are less defined. Determination of the natural history or pathophysiology associated with the lethal hematopoietic acute radiation syndrome is a significant gap in knowledge, especially when considered in the context of a nuclear weapon scenario. In this regard, the exposure is likely ill-defined, heterogenous, and nonuniform. These conditions forecast sparing of bone marrow and increased survival from the acute radiation syndrome consequent to threshold doses for the delayed effects of acute radiation exposure due to marrow sparing, medical management, and use of approved medical countermeasures. The intent herein is to provide a composite natural history of the pathophysiology concomitant with the evolution of the potentially lethal hematopoietic acute radiation syndrome derived from studies that focused on total body irradiation and partial body irradiation with bone marrow sparing. The marked differential in estimatedLD50/60from 7.5 Gy to 10.88 Gy for the total body irradiation and partial body irradiation with 5% bone marrow sparing models, respectively, provided a clear distinction between the attendant multiple organ injury and natural history of the two models that included medical management. Total body irradiation was focused on equivalentLD50/60exposures. The 10 Gy and 11 Gy partial body with 5% bone marrow sparing exposures bracketed theLD50/60(10.88 Gy). The incidence, progression, and duration of multiple organ injury was described for each exposure protocol within the hematopoietic acute radiation syndrome. The higher threshold doses for the partial body irradiation with bone marrow sparing protocol induced a marked degree of multiple organ injury to include lethal gastrointestinal acute radiation syndrome, prolonged crypt loss and mucosal damage, immune suppression, acute kidney injury, body weight loss, and added clinical comorbidities that defined a complex timeline of organ injury through the acute hematopoietic acute radiation syndrome. The natural history of the acute radiation syndrome presents a 60 d time segment of multi-organ sequelae that is concomitant with the latent period or time to onset of the evolving multi-organ injury of the delayed effects of acute radiation exposure.

Keywords: reviews; laboratory animals; mortality; radiation damage

Health Phys. 121(4):282-303; 2021

AcuteRadiationEffects,theH-ARSintheNon-humanPrimate:AReviewandNewDatafortheCynomolgusMacaquewithReferencetotheRhesusMacaque

Ann M. Farese1, Michel Drouet2, Francis Herodin3, Jean-Marc Bertho4, Karla D. Thrall5, Simon Authier6, Melanie Doyle-Eisele7, Thomas J. MacVittie1

(1.University of Maryland, School of Medicine, Department of Radiation Oncology, Baltimore;

2.Armed Forces Biomedical Research Institute, France, Department of Radiobiology, Brétigny-sur-Orge, France;

3.Ministère de la Défense, Paris, France;

4.Institute of Radiation Protection and Nuclear Safety (IRSN), 31 avenue de la division Leclerc, 92260, Fontenay-aux-Roses, France; Present address: French Nuclear Safety Authority (ASN), 15 rue Louis Lejeune 92540 Montrouge, France;

5.Consulting, LLC; 7216 Ricky Road, Pasco, WA 99301;

6.Charles River, 445 Armand Frappier, Laval, QC, Canada, H7V 4B3;

7.Lovelace Biomedical Research Institute, Laboratory Animal Sciences (Life Sciences), Albuquerque, NM)

Abstract:Medical countermeasure development under the US Food and Drug Administration animal rule requires validated animal models of acute radiation effects. The key large animal model is the non-human primate, rhesus macaque. To date, only the rhesus macaque has been used for both critical supportive data and pivotal efficacy trials seeking US Food and Drug Administration approval. The potential for use of the rhesus for other high priority studies such as vaccine development underscores the need to identify another non-human primate model to account for the current lack of rhesus for medical countermeasure development. The cynomolgus macaque,Macacafascicularis, has an existing database of medical countermeasure development against the hematopoietic acute radiation syndrome, as well as the use of radiation exposure protocols that mimic the likely nonuniform and heterogenous exposure consequent to a nuclear terrorist event. The review herein describes published studies of adult male cynomolgus macaques that used two exposure protocols—unilateral, nonuniform total-body irradiation and partial-body irradiation with bone marrow sparing—with the administration of subject-based medical management to assess mitigation against the hematopoietic acute radiation syndrome. These studies assessed the efficacy of cytokine combinations and cell-based therapy to mitigate acute radiation-induced myelosuppression. Both therapeutics were shown to mitigate the myelosuppression of the hematopoietic acute radiation syndrome. Additional studies being presented herein further defined the dose-dependent hematopoietic acute radiation syndrome of cynomolgus and rhesus macaques and a differential dose-dependent effect with young male and female cynomolgus macaques. The database supports the investigation of the cynomolgus macaque as a comparable non-human primate for efficacy testing under the US Food and Drug Administration animal rule. Critical gaps in knowledge required to validate the models and exposure protocols are also identified.

Keywords: exposure； radiation; gamma radiation; laboratory animals; radiation effects

Health Phys. 121(4):304-330; 2021

AEOL10150AlleviatesRadiation-inducedInnateImmuneResponsesinNon-humanPrimateLungTissue

Wanchang Cui1,2,3, Pei Zhang2, Kim G. Hankey2, Mang Xiao1, Ann M. Farese2, Thomas J. MacVittie2

(1.Armed Forces Radiobiology Research Institute, Uniformed Services University of the Health Sciences, Bethesda, MD 20889;

2.Department of Radiation Oncology, University of Maryland School of Medicine, Baltimore, MD 21201;

3.The Henry M. Jackson Foundation for the Advancement of Military Medicine, Bethesda, MD 20817)

Abstract:To study the molecular and cellular mechanisms of radiation-induced lung injury (RILI) in a non-human primate model, Rhesus macaques were irradiated with lethal doses of radiation to the whole thorax. A subset of the irradiated animals was treated with AEOL 10150, a potent catalytic scavenger of reactive oxygen and nitrogen species. Lung tissues were collected at necropsy for molecular and immunohistochemical (IHC) studies. Microarray expression profiling in the irradiated lung tissues identified differentially expressed genes (DEGs) and pathways important in innate immunity. The elevated expression of cytokines (CCL2, CCL11, IL-8), complement factors (CFB, C3), apoptosis-related molecules (p53, PTEN, Bax, p21, MDM2, c-Caspase 3), and adhesion molecules (fibronectin, integrin β6, ICAM-1) were further studied using real-time PCR, Western blot, or IHC. Oxidative stress and pulmonary inflammatory cell infiltration were increased in the irradiated lungs. Treatment with AEOL 10150 significantly decreased oxidative stress and monocyte/macrophage infiltration. Cytokine/chemokine-induced excessive innate immune response after thoracic irradiation plays an important role in RILI. To our knowledge, this is the first study to highlight the role of cytokine/chemokine-induced innate immune responses in radiation-induced pulmonary toxicity in a NHP model.

Keywords: health effects; lungs; human; radiation damage; radiation； biology

Health Phys. 121(4):331-344; 2021

AcuteProteomicChangesinNon-humanPrimateKidneyafterPartial-bodyRadiationwithMinimalBoneMarrowSparing

Weiliang Huang1, Jianshi Yu1, Ann M. Farese2, Thomas J. MacVittie2, Maureen A. Kane1

(1.University of Maryland, School of Pharmacy, Department of Pharmaceutical Sciences, Baltimore, MD;

2.University of Maryland, School of Medicine, Department of Radiation Oncology, Baltimore, MD 21201)

Abstract:Near total body exposure to high-dose ionizing radiation results in organ-specific sequelae, including acute radiation syndromes and delayed effects of acute radiation exposure. Among these sequelae are acute kidney injury and chronic kidney injury. Reports that neither oxidative stress nor inflammation are dominant mechanisms defining radiation nephropathy inspired an unbiased, discovery-based proteomic interrogation in order to identify mechanistic pathways of injury. We quantitatively profiled the proteome of kidney from non-human primates following 12 Gy partial body irradiation with 2.5% bone marrow sparing over a time period of 3 wk. Kidney was analyzed by liquid chromatography-tandem mass spectrometry. Out of the 3,432 unique proteins that were identified, we found that 265 proteins showed significant and consistent responses across at least three time points post-irradiation, of which 230 proteins showed strong upregulation while 35 proteins showed downregulation. Bioinformatics analysis revealed significant pathway and upstream regulator perturbations post-high dose irradiation and shed light on underlying mechanisms of radiation damage. These data will be useful for a greater understanding of the molecular mechanisms of injury in well-characterized animal models of partial body irradiation with minimal bone marrow sparing. These data may be potentially useful in the future development of medical countermeasures.

Keywords: biological indicators; kidney; radiation damage; radiation； ionizing

Health Phys. 121(4):345-351; 2021

Multi-omicAnalysisofNon-humanPrimateHeartafterPartial-bodyRadiationwithMinimalBoneMarrowSparing

Stephanie Zalesak-Kravec1?, Weiliang Huang1?, Pengcheng Wang1?, Jianshi Yu1, Tian Liu1, Amy E. Defnet1, Alexander R. Moise3, Ann M. Farese2, Thomas J. MacVittie2, Maureen A. Kane1

(1.University of Maryland, School of Pharmacy, Department of Pharmaceutical Sciences, Baltimore, MD;

2.University of Maryland, School of Medicine, Department of Radiation Oncology, Baltimore, MD;

3.Medical Sciences Division, Northern Ontario School of Medicine, Sudbury, ON, Canada; Departments of Chemistry and Biochemistry, and Biology and Biomolecular Sciences Program, Laurentian University, Sudbury, ON, Canada)

Abstract:High-dose radiation exposure results in hematopoietic and gastrointestinal acute radiation syndromes followed by delayed effects of acute radiation exposure, which encompasses multiple organs, including heart, kidney, and lung. Here we sought to further characterize the natural history of radiation-induced heart injury via determination of differential protein and metabolite expression in the heart. We quantitatively profiled the proteome and metabolome of left and right ventricle from non-human primates following 12 Gy partial body irradiation with 2.5% bone marrow sparing over a time period of 3 wk. Global proteome profiling identified more than 2,200 unique proteins, with 220 and 286 in the left and right ventricles, respectively, showing significant responses across at least three time points compared to baseline levels. High-throughput targeted metabolomics analyzed a total of 229 metabolites and metabolite combinations, with 18 and 22 in the left and right ventricles, respectively, showing significant responses compared to baseline levels. Bioinformatic analysis performed on metabolomic and proteomic data revealed pathways related to inflammation, energy metabolism, and myocardial remodeling were dysregulated. Additionally, we observed dysregulation of the retinoid homeostasis pathway, including significant post-radiation decreases in retinoic acid, an active metabolite of vitamin A. Significant differences between left and right ventricles in the pathology of radiation-induced injury were identified. This multi-omic study characterizes the natural history and molecular mechanisms of radiation-induced heart injury in NHP exposed to PBI with minimal bone marrow sparing.

Keywords: biological indicators; health effects; human organs; radiation damage

Health Phys. 121(4):352-371; 2021

ComplementaryLipidomic,Proteomic,andMassSpectrometryImagingApproachtotheCharacterizationoftheAcuteEffectsofRadiationintheNon-humanPrimateMesentericLymphNodeafterPartial-bodyIrradiationwithMinimalBoneMarrowSparing

Ludovic Muller1?, Weiliang Huang1?, Jace W. Jones1?, Ann M. Farese2, Thomas J. MacVittie2, Maureen A. Kane1

(1.University of Maryland, School of Pharmacy, Department of Pharmaceutical Sciences, Baltimore, MD,

2.University of Maryland, School of Medicine, Department of Radiation Oncology, Baltimore, MD 21201)

Abstract:Radiation sequelae is complex and characterized by multiple pathologies, which occur over time and nonuniformly throughout different organs. The study of the mesenteric lymph node (MLN) due to its importance in the gastrointestinal system is of particular interest. Other studies have shown an immediate post-irradiation reduction in cellularity due to the known effects of irradiation on lymphoid cell populations, but the molecular and functional mechanisms that lead to these cellular alterations remain limited. In this work, we show the use of lipidomic, proteomic, and mass spectrometry imaging in the characterization of the effects of acute radiation exposure on the MLN at different time points after ionizing radiation (IR) from 4 d to 21 d after 12 Gy partial body irradiation with 2.5% bone marrow sparing. The combined analyses showed a dysregulation of the lipid and protein composition in the MLN after IR. Protein expression was affected in numerous pathways, including pathways regulating lipids such as LXR/RXR activation and acute phase response. Lipid distribution and abundance was also affected by IR in the MLN, including an accumulation of triacylglycerides, a decrease in polyunsaturated glycerophospholipids, and changes in polyunsaturated fatty acids. Those changes were observed as early as 4 d after IR and were more pronounced for lipids with a higher concentration in the nodules and the medulla of the MLN. These results provide molecular insight into the MLN that can inform on injury mechanism in a non-human primate model of the acute radiation syndrome of the gastrointestinal tract. Those findings may contribute to the identification of therapeutic targets and the development of new medical countermeasures.

Keywords: biological indicators; health effects; radiation damage; radiation； ionizing

Health Phys. 121(4):372-383; 2021

AcuteProteomicChangesinLungafterRadiation:TowardIdentifyingInitiatingEventsofDelayedEffectsofAcuteRadiationExposureinNon-humanPrimateafterPartialBodyIrradiationwithMinimalBoneMarrowSparing

Weiliang Huang1, Jianshi Yu1, Tian Liu1, Amy E. Defnet1, Stephanie Zalesak-Kravec1, Ann M. Farese2, Thomas J. MacVittie2, Maureen A. Kane1

(1.University of Maryland, School of Pharmacy, Department of Pharmaceutical Sciences, Baltimore, MD;

2.University of Maryland, School of Medicine, Department of Radiation Oncology, Baltimore, MD 21201)

Abstract:Radiation-induced lung injury is a delayed effect of acute radiation exposure resulting in pulmonary pneumonitis and fibrosis. Molecular mechanisms that lead to radiation-induced lung injury remain incompletely understood. Using a non-human primate model of partial body irradiation with minimal bone marrow sparing, lung was analyzed from animals irradiated with 12 Gy at timepoints every 4 d up to 21 d after irradiation and compared to non-irradiated (sham) controls. Tryptic digests of lung tissues were analyzed by liquid chromatography-tandem mass spectrometry followed by pathway analysis. Out of the 3,101 unique proteins that were identified, we found that 252 proteins showed significant and consistent responses across at least three time points post-irradiation, of which 215 proteins showed strong up-regulation while 37 proteins showed down-regulation. Canonical pathways affected by irradiation, changes in proteins that serve as upstream regulators, and proteins involved in key processes including inflammation, fibrosis, and retinoic acid signaling were identified. The proteomic profiling of lung conducted here represents an untargeted systems biology approach to identify acute molecular events in the non-human primate lung that could potentially be initiating events for radiation-induced lung injury.

Keywords: biological indicators; lungs； human; radiation damage; radiation； ionizing

Health Phys. 121(4):384-394; 2021

MetabolomicsofMultiorganRadiationInjuryinNon-humanPrimateModelRevealsSystem-wideMetabolicPerturbations

Praveen Kumar1?, Pengcheng Wang1?, Ann M. Farese2, Thomas J. MacVittie2, Maureen A. Kane1

(1.University of Maryland, School of Pharmacy, Department of Pharmaceutical Sciences, Baltimore, MD, 21201;

2.University of Maryland, School of Medicine, Department of Radiation Oncology, Baltimore, MD 21201)

Abstract:Exposure to ionizing radiation following a nuclear or radiological incident results in potential acute radiation syndromes causing sequelae of multi-organ injury in a dose- and time-dependent manner. Currently, medical countermeasures against radiation injury are limited, and no biomarkers have been approved by regulatory authorities. Identification of circulating plasma biomarkers indicative of radiation injury can be useful for early triage and injury assessment and in the development of novel therapies (medical countermeasures). Aims of this study are to (1) identify metabolites and lipids with consensus signatures that can inform on mechanism of injury in radiation-induced multi-organ injury and (2) identify plasma biomarkers in non-human primate (NHP) that correlate with tissues (kidney, liver, lung, left and right heart, jejunum) indicative of radiation injury, assessing samples collected over 3 wk post-exposure to 12 Gy partial body irradiation with 2.5% bone marrow sparing. About 180 plasma and tissue metabolites and lipids were quantified through Biocrates AbsoluteIDQ p180 kit using liquid chromatography and mass spectrometry. System-wide perturbations of specific metabolites and lipid levels and pathway alterations were identified. Citrulline, Serotonin, PC ae 38∶2, PC ae 36∶2, and sum of branched chain amino acids were identified as potential biomarkers of radiation injury. Pathway analysis revealed consistent changes in fatty acid oxidation and metabolism and perturbations in multiple other pathways.

Keywords: biological indicators; health effects; radiation damage; radiation； ionizing

Health Phys. 121(4):395-405; 2021

EffectofRadiationontheEssentialNutrientHomeostasisandSignalingofRetinoidsinaNon-humanPrimateModelwithMinimalBoneMarrowSparing

Jianshi Yu1?, Weiliang Huang1?, Tian Liu1, Amy E. Defnet1, Stephanie Zalesak-Kravec1, Ann M. Farese2, Thomas J. MacVittie2, Maureen A. Kane1

(1.University of Maryland, School of Pharmacy, Department of Pharmaceutical Sciences, Baltimore, MD;

2.University of Maryland, School of Medicine, Department of Radiation Oncology, Baltimore, MD 21201)

Abstract:High-dose radiation exposure results in hematopoietic (H) and gastrointestinal (GI) acute radiation syndromes (ARS) followed by delayed effects of acute radiation exposure (DEARE), which include damage to lung, heart, and GI. Whereas DEARE includes inflammation and fibrosis in multiple tissues, the molecular mechanisms contributing to inflammation and to the development of fibrosis remain incompletely understood. Reports that radiation dysregulates retinoids and proteins within the retinoid pathway indicate that radiation disrupts essential nutrient homeostasis. An active metabolite of vitamin A, retinoic acid (RA), is a master regulator of cell proliferation, differentiation, and apoptosis roles in inflammatory signaling and the development of fibrosis. As facets of inflammation and fibrosis are regulated by RA, we surveyed radiation-induced changes in retinoids as well as proteins related to and targets of the retinoid pathway in the non-human primate after high dose radiation with minimal bone marrow sparing (12 Gy PBI/BM2.5). Retinoic acid was decreased in plasma as well as in lung, heart, and jejunum over time, indicating a global disruption of RA homeostasis after IR. A number of proteins associated with fibrosis and with RA were significantly altered after radiation. Together these data indicate that a local deficiency of endogenous RA presents a permissive environment for fibrotic transformation.

Keywords: biological indicators; radiation damage; radiation; ionizing; partial body irradiation

Health Phys. 121(4):406-418; 2021

RatModelsofPartial-bodyIrradiationwithBoneMarrow-sparing(Leg-outPBI)DesignedforFDAApprovalofCountermeasuresforMitigationofAcuteandDelayedInjuriesbyRadiation

Brian L. Fish1, Thomas J. MacVittie4, Feng Gao1, Jayashree Narayanan1, Tracy Gasperetti1, Dana Scholler1, Yuri Sheinin2, Heather A. Himburg1, Barry Hart3, Meetha Medhora1

(1.Department of Radiation Oncology, Medical College of Wisconsin, 8701 Watertown Plank Road, Milwaukee, WI 53226;

2.Innovation Pathways, Palo Alto, CA 94301;

3.Department of Pathology, Medical College of Wisconsin, 9200 Watertown Plank Road, Milwaukee, WI 53226;

4.Department of Radiation Oncology, University of Maryland, School of Medicine, Baltimore, MD 21201)

Abstract:The goal of this study was to develop rat models of partial body irradiation with bone-marrow sparing (leg-out PBI) to test medical countermeasures (MCM) of both acute radiation syndrome (ARS) and delayed effects of acute radiation exposure (DEARE) under the FDA animal rule. The leg-out PBI models were developed in female and male WAG/RijCmcr rats at doses of 12.5-14.5 Gy. Rats received supportive care consisting of fluids and antibiotics. Gastrointestinal ARS (GI-ARS) was assessed by lethality to d 7 and diarrhea scoring to d 10. Differential blood counts were analyzed between d 1-42 for the natural history of hematopoietic ARS (H-ARS). Lethality and breathing intervals (BI) were measured between d 28-110 to assess delayed injury to the lung (L-DEARE). Kidney injury (K-DEARE) was evaluated by measuring elevation of blood urea nitrogen (BUN) between d 90-180. TheLD50/30, including both lethality from GI-ARS and H-ARS, for female and male rats are 14.0 Gy and 13.5 Gy, respectively, while theLD50/7for only GI-ARS are 14.3 Gy and 13.6 Gy, respectively. The all-cause mortalities, including ARS and L-DEARE, through 120 d (LD50/120) are 13.5 Gy and 12.9 Gy, respectively. Secondary end points confirmed occurrence of four distinct sequelae representing GI, hematopoietic, lung, and kidney toxicities after leg-out PBI. Adult rat models of leg-out PBI showed the acute and long-term sequelae of radiation damage that has been reported in human radiation exposure case studies. Sex-specific differences were observed in the DRR between females and males. These rat models are among the most useful for the development and approval of countermeasures for mitigation of radiation injuries under the FDA animal rule.

Keywords: biological indicators; health effects; radiation effects; rat

Health Phys. 121(4):419-433; 2021

EvaluationofRadiation-inducedPleuralEffusionsafterRadiotherapytoSupportDevelopmentofAnimalModelsofRadiationPneumonitis

Masooma Aqeel1, Meetha Medhora2,3,4, Elizabeth Gore3,4, Jenna Borkenhagen3, Slade Klawikowski3, Daniel Eastwood5, Anjishnu Banerjee5, Elizabeth R. Jacobs2,4

(1.Current Affiliation: Section of Pulmonary & Critical Care Medicine, Department of Medicine, Aga Khan University, Karachi, Pakistan. Formerly at Division of Pulmonary Medicine, Department of Medicine, Froedtert Hospital & Medical College of Wisconsin, Milwaukee, WI;

2.Division of Pulmonary Medicine, Department of Medicine, Froedtert Hospital & Medical College of Wisconsin, Milwaukee, WI;

3.Department of Radiation Oncology, Froedtert Hospital & Medical College of Wisconsin, Milwaukee, WI;

4.Research Service, Department of Veteran’s Affairs, Clement J. Zablocki VA Medical Center, Milwaukee, WI;

5.Department of Biostatistics, Medical College of Wisconsin, Milwaukee, WI)

Abstract:Not all animal models develop radiation-induced pleural effusions (RIPEs) as a form of radiation-induced lung injury (RILI). Such effusions are also not well characterized in humans. The purpose of this study is to identify occurrences of RIPE in humans, provide justification for development of relevant animal models, and further characterize its risk factors in cancer patients. We also aim to identify dose thresholds for cardiopulmonary toxicity in humans to shed light on possible pathogenic mechanisms for RIPEs. We carried out a retrospective review of medical records of 96 cancer patients receiving thoracic irradiation (TRT) at our institution. Fifty-three (53%) patients developed a new pleural effusion post TRT; 18 (19%) had RIPE; and 67% developed RIPE ipsilateral to the site irradiated. None developed “contralateral only” effusions. Median time to development was 6 mo (IQR; 4-8 mo). Of 18, 8 patients (44%) had concomitant asymptomatic (radiographic only) or symptomatic radiation pneumonitis and pericardial effusion. Dosimetric factors, including combined and ipsilateral mean lung dose (MLD), were significantly associated with increased risk of RIPE. Angiotensin converting enzyme inhibition, steroids, or concurrent chemotherapy did not modify incidence of RIPE. Our results substantiate the occurrence and incidence of RIPEs in humans. In cancer patients, a median time to development of effusions around 6 mo also supports the onset of RIPEs concurrent with radiation pneumonitis. Future work needs to include large populations of cancer survivors in whom delayed RIPEs can be tracked and correlated with cardiovascular changes in the context of injury to multiple organs.

Keywords: cancer; health effects; lungs, human; radiation damage

Health Phys. 121(4):434-443; 2021