Computer aided therapeutic tripeptide design, in alleviating the pathogenic proclivities of nocuous α-synuclein fibrils.

Parkinson's disorder (PD) exacerbates neuronal degeneration of motor nerves, thereby effectuating uncoordinated movements and tremors. Aberrant alpha-synuclein (α-syn) is culpable of triggering PD, wherein cytotoxic amyloid aggregates of α-syn get deposited in motor neurons to instigate neuro-degeneration. Amyloid aggregates, typically rich in beta sheets are cardinal targets to mitigate their neurotoxic effects. In this analysis, owing to their interaction specificity, we formulated an efficacious tripeptide out of the aggregation-prone region of α-syn protein. With the help of a proficient computational pipeline, systematic peptide shortening and an adept molecular simulation platform, we formulated a tripeptide, VAV from α-syn structure based hexapeptide KISVRV. Indeed, the VAV tripeptide was able to effectively mitigate the α-syn amyloid fibrils' dynamic rate of beta-sheet formation. Additional trajectory analyses of the VAV- α-syn complex indicated that, upon its dynamic interaction, VAV efficiently altered the distinct pathogenic structural dynamics of α-syn, further advocating its potential in alleviating aberrant α-syn's amyloidogenic proclivities. Consistent findings from various computational analyses have led us to surmise that VAV could potentially re-alter the pathogenic conformational orientation of α-syn, essential to mitigate its cytotoxicity. Hence, VAV tripeptide could be an efficacious therapeutic candidate to efficiently ameliorate aberrant α-syn amyloid mediated neurotoxicity, eventually attenuating the nocuous effects of PD.Communicated by Ramaswamy H. Sarma.

Probing the polyphenolic flavonoid, morin as a highly efficacious inhibitor against amyloid(A4V) mutant SOD1 in fatal amyotrophic lateral sclerosis.

Deposition of misfolded protein aggregates in key areas of human brain is the quintessential trait of various pertinent neurodegenerative disorders including amyotrophic lateral sclerosis (ALS). Genetic point mutations in Cu/Zn superoxide dismutase (SOD1) are found to be the most important contributing factor behind familial ALS. Especially, single nucleotide polymorphism (SNP) A4V is the most nocuous since it substantially decreases life expectancy of patients. Besides, the use of naturally occurring polyphenolic flavonoids is profoundly being advocated for palliating amyloidogenic behavior of proteopathic proteins. In the present analysis, through proficient computational tools, we have attempted to ascertain a pharmacodynamically promising flavonoid compound that effectively curbs the pathogenic behavior of A4V SOD1 mutant. Initial screening of flavonoids that exhibit potency against amyloids identified morin, myricetin and epigallocatechin gallate as promising leads. Further, with the help of feasible and yet adept protein-ligand interaction studies and stalwart molecular simulation analyses, we were able to observe that aforementioned flavonoids were able to considerably divert mutant A4V SOD1 from its distinct pathogenic behavior. Among which, morin showed the most curative potential against A4V SOD1. Therefore, morin holds a great therapeutic potential in contriving highly efficacious inhibitors in mitigating fatal and insuperable ALS.

Probing the competitive inhibitor efficacy of frog-skin alpha helical AMPs identified against ACE2 binding to SARS-CoV-2 S1 spike protein as therapeutic scaffold to prevent COVID-19.

In COVID-19 infection, the SARS-CoV-2 spike protein S1 interacts to the ACE2 receptor of human host, instigating the viral infection. To examine the competitive inhibitor efficacy of broad spectrum alpha helical AMPs extracted from frog skin, a comparative study of intermolecular interactions between viral S1 and AMPs was performed relative to S1-ACE2p interactions. The ACE2 binding region with S1 was extracted as ACE2p from the complex for ease of computation. Surprisingly, the Spike-Dermaseptin-S9 complex had more intermolecular interactions than the other peptide complexes and importantly, the S1-ACE2p complex. We observed how atomic displacements in docked complexes impacted structural integrity of a receptor-binding domain in S1 through conformational sampling analysis. Notably, this geometry-based sampling approach confers the robust interactions that endure in S1-Dermaseptin-S9 complex, demonstrating its conformational transition. Additionally, QM calculations revealed that the global hardness to resist chemical perturbations was found more in Dermaseptin-S9 compared to ACE2p. Moreover, the conventional MD through PCA and the torsional angle analyses indicated that Dermaseptin-S9 altered the conformations of S1 considerably. Our analysis further revealed the high structural stability of S1-Dermaseptin-S9 complex and particularly, the trajectory analysis of the secondary structural elements established the alpha helical conformations to be retained in S1-Dermaseptin-S9 complex, as substantiated by SMD results. In conclusion, the functional dynamics proved to be significant for viral Spike S1 and Dermaseptin-S9 peptide when compared to ACE2p complex. Hence, Dermaseptin-S9 peptide inhibitor could be a strong candidate for therapeutic scaffold to prevent infection of SARS-CoV-2.

Molecular simulation probes the potency of resveratrol in regulating the toxic aggregation of mutant V30M TTR fibrils in Transthyretin mediated amyloidosis.

Transthyretin (TTR) mediated amyloidosis is a highly ruinous illness that affects various organs by aggravating the deposition of misfolded or mutated TTR protein aggregates in tissues. Hence, hindering the formation of TTR amyloid aggregates could be a key strategy in finding an effective cure towards the aggravating disorder. In this analysis, we examined the subversive nature of point mutation, V30M, in TTR that promotes amyloidogenicity using discrete molecular dynamics (DMD) simulations. Besides, we probed the association of naturally occurring polyphenols: EGCG (a proven anti TTR aggregation agent as positive control), resveratrol and curcumin in mitigating the pathogenic repercussions of mutant TTR. Results from the computational studies endorsed that the resveratrol constitutes a restorative potential to subjugate TTR mediated amyloidosis, besides EGCG. Hence, this study could be a reminiscent aspect in understanding the inhibitory role of key polyphenols against the mutant TTR aggregates, which could be an aid towards structure-based drug design in the upcoming research era on familial amyloid disorders.

Molecular simulation unravels the amyloidogenic misfolding of nascent ApoA1 protein, driven by deleterious point mutations occurring in between 170-178 hotspot region.

Protein ApoA1 is extensively studied for its role in lipid metabolism. Its seedy dark side of amyloid formulation remains relatively understudied yet. Due to genetic mutations, the protein pathologically misshapes into its amyloid form that gets accumulated in various organs, including the heart. To contrive effective therapeutics against this debilitating congenital disorder, it is imperative to comprehend the structural ramifications induced by mutations in APoA1's dynamic conformation. Till now, several point mutations have been implicated in ApoA1's amyloidosis, although only a handful has been examined considerably. Especially, the single nucleotide polymorphisms (SNPs) that occur in-between 170-178 mutation hotspot site of APoA1 needs to be investigated, since most of them are culpable of amyloid deposition in the heart. To that effect, in the present study, we have computationally quantified and studied the ApoA1's biomolecular modifications fostered by SNPs in the 170-178 mutation hotspot. Findings from discrete molecular dynamics simulation studies indicate that the SNPs have noticeably steered the ApoA1's behaviour from its native structural dynamics. Analysis of protein's secondary structural changes exhibits a considerable change upon mutations. Further, subjecting the protein structures to simulated thermal denaturation shows increased resistance to denaturation among mutants when compared to native. Further, normal mode analysis of protein's dynamic motion also shows discrepancy in its dynamic structural change upon SNP. These structural digressions induced by SNPs can very well be the biomolecular incendiary that drives ApoA1 into its amyloidogenesis. And, understanding these structural modifications initiates a better understanding of SNP's amyloidogenic pathology on APoA1.Communicated by Ramaswamy H. Sarma.

Young adults' experiences with loss and grief during COVID-19.

In the wake of the COVID-19 pandemic, people are experiencing unprecedented cumulative loss and grief. Guided by life course theory, we used inductive qualitative analysis and explored young adults' (N = 86) written accounts of their earliest and most significant losses associated with COVID-19, as well as their coping mechanisms. Overall, participants experienced substantial loss, especially losses related to their education and social life/events. We discuss five subthemes related to approaches to coping and five subthemes related to barriers to coping. Our findings are relevant to informing strategies that support adapting to significant loss in early adulthood, beyond the pandemic.

Alpha-Synuclein Aggregation in Parkinson's Disease.

Parkinson's disease (PD), a neurodegenerative disorder characterized by distinct aging-independent loss of dopaminergic neurons in substantia nigra pars compacta (SNpc) region urging toward neuronal loss. Over the decade, various key findings from clinical perspective to molecular pathogenesis have aided in understanding the genetics with assorted genes related with PD. Subsequently, several pathways have been incriminated in the pathogenesis of PD, involving mitochondrial dysfunction, protein aggregation, and misfolding. On the other hand, the sporadic form of PD cases is found with no genetic linkage, which still remain an unanswered question? The exertion in ascertaining vulnerability factors in PD considering the genetic factors are to be further dissevered in the forthcoming decades with advancement in research studies. One of the major proponents behind the prognosis of PD is the pathogenic transmutation of aberrant alpha-synuclein protein into amyloid fibrillar structures, which actuates neurodegeneration. Alpha-synuclein, transcribed by SNCA gene is a neuroprotein found predominantly in brain. It is implicated in the modulation of synaptic vesicle transport and eventual release of neurotransmitters. Due to genetic mutations and other elusive factors, the alpha-synuclein misfolds into its amyloid form. Therefore, this review aims in briefing the molecular understanding of the alpha-synuclein associated with PD.

Unravelling the molecular effect of ocellatin-1, F1, K1 and S1, the frog-skin antimicrobial peptides to enhance its therapeutics-quantum and molecular mechanical approaches.

Ocellatin AMPs (antimicrobial peptides) are considered to be promising alternative therapeutics to conventional antibiotics. Three-dimensional (3D) structures of ocellatin-F1 with 25 residues have been reported to be potent in terms of bacterial membrane permeability. To investigate the influence of similar ocellatin peptides with 25 residues pertaining to antimicrobial effect, ocellatin-1, K1 and S1 peptides were modelled with ocellatin-F1 as template. Comparative analyses between these peptides were carried out, using computational approaches. From the results of in silico toxicity profile, all peptides were found to be non-toxic with no haemolytic activity. Further sequence analysis, net charge, hydrophobicity and hydrophobic moment revealed the membrane permeable efficacy of ocellatin-1 peptide. Besides, the investigation of peptide electronic structures through density functional theory and quantum chemical (HOMO and LUMO) calculations predicted ocellatin-1 to be a suitable peptide, which can be used as a scaffold for therapeutics. Furthermore, the determination of structural contours such as RMSD, RMSF and Rg through trajectory analysis revealed that ocellatin-1 exhibited strong structural stability. In addition, the trajectory analysis of elements of secondary structure illustrated the alpha helical conformations to be retained in all peptides, except ocellatin-1. On the aforementioned grounds, ocellatin-1 was found to possess the important role of peptide penetration of the bacterial membrane. This study becomes significant, since it is the first time where the structural importance of ocellatin peptides were explored in detail and the therapeutic potential of ocellatin-1 as a peptide-based antimicrobial drug have been theoretically revealed.

Plasmodium falciparum metacaspase-2 capture its natural substrate in a non-canonical way.

Programmed cell death (PCD) is a multi-step process initiated by a set of proteases, which interacts and cleaves diverse proteins, thus modulating their biochemical and cellular functions. In metazoans, PCD is mediated by proteolytic enzymes called caspases, which triggered cell death by proteolysis of human Tudor staphylococcus nuclease (TSN). Non-metazoans lack a close homologue of caspases but possess an ancestral family of cysteine proteases termed 'metacaspases'. Studies supported that metacaspases are involved in PCD, but their natural substrates remain unknown. In this study, we performed the Plasmodium falciparum TSN (PfTSN) cleavage assay using wild and selected mutants of P. falciparum metacaspases-2 (PfMCA-2) in vitro and in vivo. Interestingly, PfMCA-2, cleaved a phylogenetically conserved protein, PfTSN at multiple sites. Deletion or substitution mutation in key interacting residues at the active site, Cys157 and His205 of PfMCA-2, impaired its enzymatic activity with the artificial substrate, z-GRR-AMC. However, the mutant Tyr224A did not affect the activity with z-GRR-AMC but abolished the cleavage of PfTSN. These results indicated that the catalytic dyad, Cys157 and His205 of PfMCA-2 was essential for its enzymatic activity with an artificial substrate, whereas Tyr224 and Cys157 residues were responsible for its interaction with the natural substrate and subsequent degradation of PfTSN. Our results suggested that MCA-2 interacts with TSN substrate in a non-canonical way using non-conserved or conformationally available residues for its binding and cleavage. In future, it would be interesting to explore how this interaction leads to the execution of PCD in the Plasmodium.

Decoding Conformational Imprint of Convoluted Molecular Interactions Between Prenylflavonoids and Aggregated Amyloid-Beta42 Peptide Causing Alzheimer's Disease.

Protein misfolding occurs due to the loss of native protein structure and adopts an abnormal structure, wherein the misfolded proteins accumulate and form aggregates, which result in the formation of amyloid fibrils that are associated with neurodegenerative diseases. Amyloid beta (Aß42) aggregation or amyloidosis is contemplated as a unique hallmark characteristic of Alzheimer's disease (AD). Due to aberrant accrual and aggregation of Aß42 in extracellular space, the formation of senile plaques is found in AD patients. These senile plaques occur usually in the cognitive and memory region of the brain, enfeebles neurodegeneration, hinders the signaling between synapse, and disrupts neuronal functioning. In recent years, herbal compounds are identified and characterized for their potential as Aß42 inhibitors. Thus, understanding their structure and molecular mechanics can provide an incredible finding in AD therapeutics. To describe the structure-based molecular studies in the rational designing of drugs against amyloid fibrils, we examined various herbal compounds that belong to prenylflavonoids. The present study characterizes the trends we identified at molecular docking studies and dynamics simulation where we observed stronger binding orientation of bavachalcone, bavachin, and neobavaisoflavone with the amyloid-beta (Aß42) fibril structure. Hence, we could postulate that these herbal compounds could be potential inhibitors of Aß42 fibrils; these anti-aggregation agents need to be considered in treating AD.

TTRMDB: A database for structural and functional analysis on the impact of SNPs over transthyretin (TTR) using bioinformatic tools.

Hereditary Transthyretin-associated amyloidosis (ATTR) is an autosomal dominant protein-folding disorder with adult-onset caused by mutation of transthyretin (TTR). TTR is characterized by extracellular deposition of amyloid, leading to loss of autonomy and finally, death. More than 100 distinct mutations in TTR gene have been reported from variable age of onset, clinical expression and penetrance data. Besides, the cure for the disease remains still obscure. Further, the prioritizing of mutations concerning the characteristic features governing the stability and pathogenicity of TTR mutant proteins remains unanswered, to date and thus, a complex state of study for researchers. Herein, we provide a full report encompassing the effects of every reported mutant model of TTR protein about the stability, functionality and pathogenicity using various computational tools. In addition, the results obtained from our study were used to create TTRMDB (Transthyretin mutant database), which could be easy access to researchers at http://vit.ac.in/ttrmdb.

A Systematic and Comprehensive Review on Disease-Causing Genes in Amyotrophic Lateral Sclerosis.

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disorder and is characterized by degeneration and axon loss from the upper motor neuron, that descends from the lower motor neuron in the brain. Over the period, assorted outcomes from medical findings, molecular pathogenesis, and structural and biophysical studies have abetted in providing thoughtful insights underlying the importance of disease-causing genes in ALS. Consequently, numerous mechanisms were proposed for the pathogenesis of ALS, considering protein mutations, aggregation, and misfolding. Besides, the answers to the majority of ALS cases that happen to be sporadic still remain obscure. The application in discovering susceptibility factors in ALS contemplating the genetic factors is to be further dissevered in the future years with innovation in research studies. Hence, this review targets in revisiting the breakthroughs on the disease-causing genes related with ALS.

Comparison of survival of stage I-III colon cancer by travel distance and hospital volume.

BACKGROUND: Previous studies have demonstrated improved outcomes at high-volume colorectal surgery centers; however, the benefit for patients who live far from such centers has not been assessed relative to local, low-volume facilities. METHODS: The 2010-2015 National Cancer Database (NCDB) was queried for patients with stage I-III colon adenocarcinoma undergoing treatment at a single center. A 'local, low-volume' cohort was constructed of 12,768 patients in the bottom quartile of travel distance at the bottom quartile of institution surgical volume and a 'travel, high-volume' cohort of 11,349 patients in the top quartile of travel distance at the top quartile of institution surgical volume. RESULTS: In unadjusted analysis, patients in the travel cohort had improved rates of positive resection margins (3.7% vs. 5.5%, p < 0.001), adequate lymph-node harvests (92% vs. 83.6%, p < 0.001), and 30- (2.2% vs. 3.9%, p < 0.001) and 90-day mortality (3.7% vs. 6.4%, p < 0.001). On multivariable logistic regression analysis adjusting for patient demographic, tumor, and facility characteristics, the cohorts demonstrated equivalent overall survival (HR: 0.972, p = 0.39), with improved secondary outcomes in the 'travel' cohort of adequate lymph-node harvesting (OR: 0.57, p < 0.001), and 30- (OR 0.79, p = 0.019) and 90-day mortality (OR 0.80, p = 0.004). CONCLUSIONS: For patients with stage I-III colon cancer, traveling to high-volume institutions compared to local, low-volume centers does not convey an overall survival benefit. However, given advantages including 30- and 90-day mortality and adequate lymph-node harvest, nuanced patient recommendations should consider both these differences and the unquantified benefits to local care, including cost, travel time, and support systems.

Rational design of linear tripeptides against the aggregation of human mutant SOD1 protein causing amyotrophic lateral sclerosis.

Formation of protein aggregation is considered a hallmark feature of various neurological diseases. Amyotrophic lateral sclerosis is one such devastating neurodegenerative disorder characterized by mutation in Cu/Zn superoxide dismutase protein (SOD1). In our study, we contemplated the most aggregated and pathogenic mutant A4V in a viewpoint of finding a therapeutic regime by inhibiting the formation of the aggregates with the aid of tripeptides since new perspectives in the field of drug design in the current era are being focused on peptide-based drugs. Reports from the experimental study have stipulated that the SOD1 derived peptide, "LSGDHCIIGRTLVVHEKADD" was found to have the inhibitory activity against aggregated SOD1 protein. Moreover, it was determined that the hexapeptide, "LSGDHC" was the key factor in inhibiting the aggregates of SOD1. Accordingly, we utilized the computerized algorithms and programs on determining the binding efficiency and inhibitory activity of hexapeptide on mutant SOD1. Following that, we incorporated a cutting-edge methodology with the use of molecular docking, affinity predictions, alanine scanning, steered molecular dynamics (SMD) and discrete molecular dynamics (DMD) in designing the de novo tripeptides, which could act against the aggregated mutant SOD1 protein. Upon examining the results from the various conformational studies, we identified that CGH had an enhanced binding affinity and inhibitory activity against the aggregated mutant SOD1 protein than other tripeptides and hexapeptide. Thus, our study could be a lead for state-of-the-art design in peptide-based drugs for doctoring the cureless ALS disorder.

Computational Investigation on Electrostatic Loop Mutants Instigating Destabilization and Aggregation on Human SOD1 Protein Causing Amyotrophic Lateral Sclerosis.

Mutations in the gene encoding Cu/Zn Superoxide Dismutase 1 (SOD1) protein are contemplated to be a protruding reason for Amyotrophic lateral sclerosis (ALS), which leads towards protein aggregation, misfolding and destabilization. Thus, we investigated the systematic action of entire mutations reported on electrostatic loop of SOD1 protein through thermodynamical and discrete molecular dynamics (DMD) studies. Accordingly, we analyzed the outcomes distinctly for screening the mutant structures having both, deleterious and destabilizing effect. Progressively, the impacts of those mutations on SOD1 were studied using DMD program. Surprisingly, our results predicted that the mutants viz., L126S, N139H and G141A to be the most destabilizing, misfolded and disease-causing compared to other mutants. Besides, the outcomes from secondary structural propensities and free energy landscapes, together assertively suggested that L126S, N139H and G141A tend to increase the formation of aggregates in SOD1 relative to other mutants. Hence, this study could provide an insight into the sprouting neurodegenerative disorder distressing the humans.

Molecular binding response of naringin and naringenin to H46R mutant SOD1 protein in combating protein aggregation using density functional theory and discrete molecular dynamics.

Amyotrophic lateral sclerosis (ALS) is a calamitous neurodegenerative disorder characterized by denervation of upper and lower motor neurons. Numerous hypotheses suggest that toxic protein misfolding and aggregation cause ALS, similar to that of other neurodegenerative diseases, such as Alzheimers and Parkinsons. Protruding causes of familial ALS are mutations in the gene encoding Cu/Zn superoxide dismutase-1 (SOD1), which decrease protein stability and endorse protein aggregation. Thus, the interference concerning aggregate formation and destabilization in SOD1 is considered to be an impending therapeutic strategy. In this work, we utilized computational chemistry methods to initially study the effect of substitution mutation, His46Arg on SOD1 protein. Further, we described the interaction of two naturally occurring polyphenol compounds, naringin and naringenin with mutant SOD1 that is regarded to hinder the protein aggregation. Subsequently, the use of quantum chemical and molecular mechanics calculations speculated that naringin had a strong binding affinity with mutant SOD1 and impeded the formation of toxic aggregates than that of naringenin. Ultimately, we could conjecture that ingesting of polyphenol-rich foods in ALS patients may be regarded to improvise their living. Moreover, the findings from our study could pave a way in the field of structure-based drug design in developing potential anti-aggregation inhibitors against incurable ALS, affecting the human population.

Bereavement and the Oregon Death with Dignity Act: How does assisted death impact grief?

This qualitative study explores bereavement experiences with an assisted death. Thematic analysis of data from interviews with 22 people who had a family member die under Oregon's Death with Dignity Act revealed the following five themes: (a) general grief reactions, (b) anticipating the death, (c) sense of control, (d) level of agreement with assisted death, and (e) grief expression and stigma. Grief from an assisted death is a varied experience, including aspects that are unique to this mode of death, and aspects that can both ease and bring challenges to the grieving process.

Quantum chemical and molecular mechanics studies on the assessment of interactions between resveratrol and mutant SOD1 (G93A) protein.

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease that has been associated with mutations in metalloenzyme superoxide dismutase (SOD1) causing protein structural destabilization and aggregation. However, the mechanistic action and the cure for the disease still remain obscure. Herein, we initially studied the conformational preferences of SOD1 protein structures upon substitution of Ala at Gly93 in comparison with that of wild type. Our results corroborated with the previous experimental studies on the aggregation and the destabilizing activity of mutant SOD1 protein G93A. On the therapeutic point of view, we computationally analyzed the influence of resveratrol, a natural polyphenol widely found in red wine on mutant SOD1 relative to wild type, using molecular docking studies. Further, FMO calculations were performed, using GAMESS to study the pair residual interaction on the wild type and mutant complex systems. Consequently, the resveratrol showed greater interaction with mutant than the wild type. Subsequently, we evaluated the conformational preferences of wild type and mutant complex systems, where the protein conformational structures of mutant that were earlier found to lose their conformational stability was regained, upon binding with resveratrol. Similar trend of results were found on the 2-D free energy landscapes of both the wild type and mutant systems. Hence, the combined biophysical and quantum chemical studies in our study supported the results of previous experimental studies, thereby stipulating an action of resveratrol on mutant SOD1 and paving a way for the design of highly potent effective inhibitors against fALS affecting the mankind.

Comparative binding of kaempferol and kaempferide on inhibiting the aggregate formation of mutant (G85R) SOD1 protein in familial amyotrophic lateral sclerosis: A quantum chemical and molecular mechanics study.

Mutation in Cu/Zn superoxide dismutase (SOD1) at position 85 from glycine to arginine was found to be a prominent cause of aggregation characterized by an increased content of ß-sheets in familial amyotrophic lateral sclerosis (fALS). Various literatures reported that natural polyphenols could act as a ß-sheet breaker and therefore, treated as a potential therapeutics against various aggregated proteins involved in neurodegenerative disorders. Through computational perspective, molecular docking, quantum chemical studies, and discrete molecular dynamics were implemented to study the binding and structural effect of natural polyphenols, kaempferol, and kaempferide on mutant SOD1. Kaempferol exhibited significant binding and greater residual energy contribution with mutant SOD1 than kaempferide. More interestingly, kaempferol was found to reduce the ß-sheet content augmenting the mutant conformational stability and flexibility relative to that of kaempferide. Hence, the inhibition of mutant SOD1 aggregation by kaempferol was explored, thereby suggesting kaempferol could act as a drug candidate for the design of the natural therapeutics against fALS. © 2018 BioFactors, 44(5):431-442, 2018.