Design, Synthesis, and Insecticidal Activity of Pyridino[1,2-a]pyrimidines Containing Indole Moeites at the 1-Position.

Research on mesoionic structures in pesticide design has gained significant attention in recent years. However, the 1-position of pyridino[1,2-a]pyrimidine is usually designed with 2-chlorothiazole, 2-chloropyridine, or cyano moieties commonly found in neonicotinoid insecticides. In order to enrich the available pharmacophore library, here, we disclose a series of new pyridino[1,2-a]pyrimidine mesoionics bearing indole-containing substituents at the 1-position. Most of these target compounds are confirmed to have good insecticidal activity against aphids through bioevaluation. In addition, a three-dimensional structure-activity relationship model is established to allow access to optimal compound F45 with an LC50 value of 2.97 mg/L. This value is comparable to the property achieved by the positive control triflumezopyrim (LC50 = 2.94 mg/L). Proteomics and molecular docking analysis suggest that compound F45 has the potential to modulate the functioning of the aphid nervous system through its interaction with neuronal nicotinic acetylcholine receptors. This study expands the existing pharmacophore library for the future development of new mesoionic insecticides based on 1-position modifications of the pyridino[1,2-a]pyrimidine scaffold.

Design, Synthesis, Antibacterial Activity, and Mechanisms of Novel Benzofuran Derivatives Containing Disulfide Moieties.

The unsatisfactory effects of conventional bactericides and antimicrobial resistance have increased the challenges in managing plant diseases caused by bacterial pests. Here, we report the successful design and synthesis of benzofuran derivatives using benzofuran as the core skeleton and splicing the disulfide moieties commonly seen in natural substances with antibacterial properties. Most of our developed benzofurans displayed remarkable antibacterial activities to frequently encountered pathogens, including Xanthomonas oryzae pv oryzae (Xoo), Xanthomonas oryzae pv oryzicola (Xoc), and Xanthomonas axonopodis pv citri (Xac). With the assistance of the three-dimensional quantitative constitutive relationship (3D-QSAR) model, the optimal compound V40 was obtained, which has better in vitro antibacterial activity with EC50 values of 0.28, 0.56, and 10.43 µg/mL against Xoo, Xoc, and Xac, respectively, than those of positive control, TC (66.41, 78.49, and 120.36 µg/mL) and allicin (8.40, 28.22, and 88.04 µg/mL). Combining the results of proteomic analysis and enzyme activity assay allows the antibacterial mechanism of V40 to be preliminarily revealed, suggesting its potential as a versatile bactericide in combating bacterial pests in the future.

Synthesis, Bioactivities, and Antibacterial Mechanism of 5-(Thioether)-N-phenyl/benzyl-1,3,4-oxadiazole-2-carboxamide/amine Derivatives.

1,3,4-Oxadiazole thioethers have shown exciting antibacterial activities; however, the current mechanism of action involving such substances against bacteria is limited to proteomics-mediated protein pathways and differentially expressed gene analysis. Herein, we report a series of novel 1,3,4-oxadiazole thioethers containing a carboxamide/amine moiety, most of which show good in vitro and in vivo bacteriostatic activities. Compounds A10 and A18 were screened through CoMFA models as optimums against Xanthomonas oryzae pv. oryzae (Xoo, EC50 values of 5.32 and 4.63 mg/L, respectively) and Xanthomonas oryzae pv. oryzicola (Xoc, EC50 values of 7.58 and 7.65 mg/L, respectively). Compound A10 was implemented in proteomic techniques and activity-based protein profiling (ABPP) analysis to elucidate the antibacterial mechanism and biochemical targets. The results indicate that A10 disrupts the growth and pathogenicity of Xoc by interfering with pathways associated with bacterial virulence, including the two-component regulation system, flagellar assembly, bacterial secretion system, quorum sensing, ABC transporters, and bacterial chemotaxis. Specifically, the translational regulator (CsrA) and the virulence regulator (Xoc3530) are two effective target proteins of A10. Knocking out the CsrA or Xoc3530 gene in Xoc results in a significant reduction in the motility and pathogenicity of the mutant strains. This study contributes available molecular entities, effective targets, and mechanism basis for the management of rice bacterial diseases.

Exploitation alters microbial community and its co-occurrence patterns in ionic rare earth mining sites.

The exploitation of ion-adsorption rare earth elements (REEs) deposits results in serious ecological and environmental problems, which has attracted much attention. However, the influences of exploitation on the prokaryotic communities and their complex interactions remain poorly understood. In the present study, bacterial and archaeal communities, as well as ammonia-oxidizing bacteria (AOB) and ammonia-oxidizing archaea (AOA), in and around REEs mining area were investigated through high throughput sequencing and quantitative polymerase chain reaction (qPCR). Our results indicated that mining soil was characterized by poor soil structure, nutrient deficiency, and high concentrations of residual REEs. Oligotrophic bacteria (e.g., Chloroflexi and Acidobacteriota) were dominant in unexploited soil and mining soil, while copiotrophic bacteria (Proteobacteria and Actinobacteriota) were more abundant in surrounding soil. Nutrient was the key factor affecting microbial variation and abundance in mining soil. The bacterial community was more sensitive to REEs, while the archaeal communities were relatively stable. As the key members for ammonia oxidation, AOA outnumbered AOB in all the soil types, and the former was significantly influenced by pH, nutrients, and TREEs in mining soil. The microbial co-occurrence network analysis demonstrated that exploitation significantly influenced topological properties, decreased the complexity, and resulted in a much unstable network, leading to a more fragile microbial ecosystem in mining areas. Notably, the abundance of keystone taxa decreased after exploitation, and oligotrophic groups (Chloroflexi) replaced copiotrophic groups (Proteobacteria and Actinobacteriota) as the key to rebuilt a co-occurrence network, suggesting potentially important roles in maintaining network stability. The current results are of great significance to the ecological risk assessment of REEs exploitation.

Design, Synthesis, Antibacterial Activity, and Mechanism of Novel Mesoionic Compounds Based on Natural Pyrazole Isolated from an Endophytic Fungus Colletotrichum gloeosporioides.

Xanthomonas oryzae pv. oryzicola (Xoo) is a type of bacteria that causes bacterial leaf blight disease in rice plants. This disease is substantially harmful, and the current prevention and control measures are facing challenges. This study has investigated the effectiveness of the control activity that the endophytic fungus NS7 fermented from Dendrobium candidum possessed against Xoo. Twenty-eight novel mesoionic compounds were designed and synthesized based on the natural compound D. These compounds displayed moderate to excellent anti-Xoo activity in vitro. Notably, compound 24 exhibited prominent anti-Xoo activity in vitro with an EC50 value of 40.3 mg/L, which was better than that of the positive control thiodiazole copper (TC)(71.2 mg/L) and the lead compound D (108.1 mg/L). In vivo pot experiments on Xoo showed that compound 24 exhibited protective and curative activities of 39.4 and 30.4%, respectively, which were better than those of TC (35.7 and 28.8%, respectively). Further, a preliminary mechanism study indicated that compound 24 could enhance the activity of defense enzymes to improve the ability for anti-Xoo. Meanwhile, compound 24 could also regulate the carbon fixation in photosynthetic organisms, which might be related to the enhanced immune function of rice. This study offers a new strategy for discovering antibacterial agents based on natural products.

Design, Synthesis, and Insecticidal Activity of Mesoionic Pyrido[1,2-a]pyrimidinone Containing Isoxazole/Isoxazoline Moiety as a Potential Insecticide.

Bean aphid (Aphis craccivora) resistance to commonly used insecticides has made controlling these pests increasingly difficult. In this study, we introduced isoxazole and isoxazoline, which possess insecticidal activity, into pyrido[1,2-a]pyrimidinone through a scaffold hopping strategy. We designed and synthesized a series of novel mesoionic compounds that exhibited a range of insecticidal activities against A. craccivora. The LC50 values of compounds E1 and E2 were 0.73 and 0.88 µg/mL, respectively, better than triflumezopyrim (LC50 = 2.43 µg/mL). Proteomics and molecular docking analyses showed that E1 might influence the A. craccivora nervous system by interacting with neuronal nicotinic acetylcholine receptors (nAChRs). This research offers a new approach to the advancement of novel mesoionic insecticides.

Design, Synthesis, and Insecticidal Activity of Novel Isoxazoline Diacylhydrazine Compounds as GABA Receptor Inhibitors.

A series of isoxazoline derivatives containing diacylhydrazine moieties were designed and synthesized as potential insecticides. Most of these derivatives exhibited good insecticidal activities against Plutella xylostella, and some compounds exhibited excellent insecticidal activities against Spodoptera frugiperda. Especially, D14 showed outstanding insecticidal activity against P. xylostella (LC50 = 0.37 µg/mL), which was superior to that of ethiprole (LC50 = 2.84 µg/mL) and tebufenozide (LC50 = 15.3 µg/mL) and similar to that of fluxametamide (LC50 = 0.30 µg/mL). Remarkably, the insecticidal activity of D14 against S. frugiperda (LC50 = 1.72 µg/mL) was superior to that of chlorantraniliprole (LC50 = 3.64 µg/mL) and tebufenozide (LC50 = 60.5 µg/mL) but lower than that of fluxametamide (LC50 = 0.14 µg/mL). The results of electrophysiological experiments, molecular docking, and proteomics experiments indicate that compound D14 acts by interfering with the γ-aminobutyric acid receptor to control pests.

Synthesis, Antibacterial Activity, and Mechanisms of Novel Indole Derivatives Containing Pyridinium Moieties.

The development of effective antibacterial agents equipped with novel action modes and unique skeletons starting from natural compounds serves as an important strategy in the modern pesticide industry. Disclosed here are a series of novel indole derivatives containing pyridinium moieties and their antibacterial activity evaluation against two prevalent phytopathogenic bacteria, Xanthomonas oryzae pv. oryzicola (Xoc) and X. oryzae pv. oryzae (Xoo). A three-dimensional (3D)-QSAR model was adopted to discover higher activity like title compounds based on the Xoc antibacterial activity of the tested compounds. Compound 43 was consequently designed, and it displayed higher antibacterial activity as expected with the half-maximal effective concentration EC50 values of 1.0 and 1.9 µg/mL for Xoo and Xoc, respectively, which were better than those of the commercial drug thiodiazole copper (TC) (72.9 and 87.5 µg/mL). Under greenhouse conditions, the results of a rice in vivo pot experiment indicated that the protective and curative activities of compound 43 against rice bacterial leaf streak (BLS) and rice bacterial blight (BLB) were 45.0 and 44.0% and 42.0 and 39.3%, respectively, which were better than those of the commercial agent thiodiazole copper (38.0 and 37.9%, 38.6 and 37.0%) as well. Scanning electron microscopy images, defense enzyme activity tests, and proteomic techniques were utilized in a preliminary mechanism study, suggesting that compound 43 shall modulate and interfere with the physiological processes and functions of pathogenic bacteria.

Pyrido [1,2-a] Pyrimidinone Mesoionic Compounds Containing Vanillin Moiety: Design, Synthesis, Antibacterial Activity, and Mechanism.

Xanthomonas oryzae pv. oryzicola (Xoo) is a plant pathogen responsible for rice bacterial blight disease that remains challenging for prevention and cure. To discover innovative and extremely potent antibacterial agents, vanillin moiety was introduced to develop a series of novel mesoionic derivatives. Compound 15 demonstrated excellent in vitro antibacterial activity against Xoo, with a 50% effective concentration value (EC50) of 27.5 µg/mL, which was superior to that of the positive control agent thiodiazole copper (97.1 µg/mL) and comparable to that of compound "A11" (17.4 µg/mL). The greenhouse pot experiment also revealed that compound 15 had 38.5% curative and 36.8% protective efficacy against rice bacterial leaf blight in vivo at 100 µg/mL, which was higher than those of thiodiazole copper (31.2 and 32.6%, respectively) and compound "A11" (29.6 and 33.2%, respectively). Compound 15 enhanced the activities of related defense enzymes, increased chlorophyll content, and promoted the resistance of rice to bacterial infection by modulating the photosynthetic pathway. This study provides a basis for the subsequent structural modification and mechanism research of mesoionic derivatives.

Discovery of Pyrido[1,2-a]pyrimidine Mesoionic Compounds Containing Benzo[b]thiophene Moiety as Potential Pesticide Candidates.

The increasing evolution of insect resistance has made it challenging for traditional insecticides to control the bean aphid (Aphis craccivora Koch). To address this pending issue, a range of pyrido[1,2-a]pyrimidine mesoionic compounds containing benzo[b]thiophene were designed and synthesized. The biological activity test results of the target compounds indicated that they had moderate to outstanding insecticidal activity against the bean aphid (Aphis craccivora) and moderate insecticidal activity against the white-backed planthopper (Sogatella furcifera). Compound L14 exhibited significant insecticidal activity against A. craccivora, with an LC50 value of 1.82 µg/mL, which was superior to triflumezopyrim (LC50 = 4.76 µg/mL). The results of enzyme activity assay showed that compound L14 had a definite inhibitory effect on ATPase. Moreover, the proteomics and docking findings of compound L14 suggested that it may act on the central nervous system of aphids and interact with nicotinic acetylcholine receptors. Therefore, compound L14 is a potentially novel insecticide candidate for further utilization.

First Discovery of Imidazo[1,2-a]pyridine Mesoionic Compounds Incorporating a Sulfonamide Moiety as Antiviral Agents.

The applications of mesoionic compounds and their analogues as agents against plant viruses remain unexplored. This was the first evaluation of the antiviral activities of mesoionic compounds on this issue. Our study involved the design and synthesis of a series of novel imidazo[1,2-a]pyridine mesoionic compounds containing a sulfonamide moiety and the assessment of their antiviral activities against potato virus Y (PVY). Compound A33 was assessed on the basis of three-dimensional quantitative structure-activity relationship (3D-QSAR) model analysis and displayed good curative, protective, and inactivating activity effects against PVY at 500 mg/L, up to 51.0, 62.0, and 82.1%, respectively, which were higher than those of commercial ningnanmycin (NNM, at 47.2, 50.1, and 81.4%). Significantly, defensive enzyme activities and proteomics results showed that compound A33 could enhance the defense response by activating the activity of defense enzymes, inducing the glycolysis/gluconeogenesis pathway of tobacco to resist PVY infection. Therefore, our study indicates that compound A33 could be applied as a potential viral inhibitor.

Coumarin Derivatives Containing Sulfonamide and Dithioacetal Moieties: Design, Synthesis, Antiviral Activity, and Mechanism.

Cucumber mosaic virus (CMV) is currently a known plant virus with the most hosts, broadest distribution, and economic hazard. To develop new antiviral drugs against this serious virus, a new range of coumarin derivatives containing sulfonamide and dithioacetal structures were designed and synthesized, and their anti-CMV activities were detected by the half-leaf dead spot method. The results of the biological activity assay showed that most of the compounds exhibited outstanding anti-CMV activity. Especially, compound C23 displayed the optimal in vivo anti-CMV activity, with an EC50 value of 128 µg/mL, which was remarkably better than that of COS (781 µg/mL) and ningnanmycin (436 µg/mL). Excitingly, we found that compound C23 could be a promising plant activator that significantly increased defense-related enzyme activities and the tobacco chlorophyll content. Furthermore, compound C23 enhanced defense responses against viral infection by inducing the abscisic acid (ABA) pathway in tobacco. This work established a basis for multifunction pesticide discovery involving mechanism of action study and structure optimization.

Design, Synthesis, and Insecticidal Activity of Novel Pyrido[1, 2-a]pyrimidinone Mesoionic Compounds Containing an Indole Moiety as Potential Acetylcholine Receptor Insecticides.

In this study, a series of novel mesoionic pyrido[1,2-a]pyrimidinone compounds containing a natural skeleton indole were designed and synthesized, and the insecticidal activities of the target compounds were tested. The results showed that the target compounds had good to excellent insecticidal activities against white-backed planthoppers (Sogatella furcifera) and bean aphids (Aphis craccivora). Among them, compound 7 showed outstanding insecticidal activities against both S. furcifera and A. craccivora, with LC50 values of 0.86 and 0.85 µg/mL, respectively. The insecticidal activity against bean aphids (A. craccivora) was superior to that of triflumezopyrim (LC50 = 3.67 µg/mL). Proteomics and quantitative real-time polymerase chain reaction (qRT-PCR) results revealed that compound 7 may interact with α1 and α7 nAChR subunits of S. furcifera. The results of enzyme activities indicated that compound 7 was an inhibitor of AChE in S. furcifera. This study provides new ideas for the discovery of new mesoionic pyrido[1,2-a]pyrimidinone insecticides.

Discovery of Pyrido[1,2-a]pyrimidinone Mesoionic Compounds Incorporating a Dithioacetal Moiety as Novel Potential Insecticidal Agents.

A series of novel mesoionic pyrido[1,2-a]pyrimidinone compounds incorporating a dithioacetal skeleton were designed and synthesized for use as insecticidal agents. The biological activity of the title compounds indicated good to excellent insecticidal activities against bean aphids (Aphis craccivora) and white-backed planthoppers (Sogatella furcifera). Compound 34 showed excellent insecticidal activity against bean aphids (A. craccivora) with an LC50 value of 2.80 µg/mL, exceeding the insecticidal activity of trifluoropyrimidine (LC50 = 4.20 µg/mL). Proteomics and molecular docking results indicated that compound 34 could act on nicotinic acetylcholine receptors. This study provides support for the application of mesoionic pyrido[1,2-a]pyrimidinone compounds containing dithioacetal as novel insecticidal agents.

Discovery of Novel Dihydrolipoamide S-Succinyltransferase Inhibitors Based on Fragment Virtual Screening.

A series of new oxadiazole sulfone derivatives containing an amide moiety was synthesized based on fragment virtual screening to screen high-efficiency antibacterial agents for rice bacterial diseases. All target compounds showed greater bactericidal activity than commercial bactericides. 3-(4-fluorophenyl)-N-((5-(methylsulfonyl)-1,3,4-oxadiazol-2-yl)methyl)acrylamide (10) showed excellent antibacterial activity against Xanthomonas oryzae pv. oryzae and Xanthomonas oryzae pv. oryzicola, with EC50 values of 0.36 and 0.53 mg/L, respectively, which were superior to thiodiazole copper (113.38 and 131.54 mg/L) and bismerthiazol (83.07 and 105.90 mg/L). The protective activity of compound 10 against rice bacterial leaf blight and rice bacterial leaf streak was 43.2% and 53.6%, respectively, which was superior to that of JHXJZ (34.1% and 26.4%) and thiodiazole copper (33.0% and 30.2%). The curative activity of compound 10 against rice bacterial leaf blight and rice bacterial leaf streak was 44.5% and 51.7%, respectively, which was superior to that of JHXJZ (32.6% and 24.4%) and thiodiazole copper (27.1% and 28.6%). Moreover, compound 10 might inhibit the growth of Xanthomonas oryzae pv. oryzae and Xanthomonas oryzae pv. oryzicola by affecting the extracellular polysaccharides, destroying cell membranes, and inhibiting the enzyme activity of dihydrolipoamide S-succinyltransferase.

Synthesis of trans- methyl ferulate bearing an oxadiazole ether as potential activators for controlling plant virus.

A series of new ferulic acid derivatives bearing an oxadiazole ether was synthesized by introducing a structure of oxadiazole into trans-ferulic acid via an ether linkage. The synthesized target compounds were evaluated in vivo for their anti-TMV (tobacco mosaic virus) activity, which indicated that some synthesized compounds displayed strong activity for controlling TMV. For protective activity, compounds 6f and 6h had the most activities of 65% and 69.8% at 500 mg L-1, respectively. Compounds 6a, 6b, 6e, 6f and 6h showed > 60% curative activities at 500 mg L-1. Preliminary proteomics analysis showed that compound 6h could regulate the phenylpropanoid biosynthesis pathway and chloroplast function. These results indicated that synthesized novel ferulic acid derivatives could be used for controlling TMV.

Facile Synthesis of Novel Vanillin Derivatives Incorporating a Bis(2-hydroxyethyl)dithhioacetal Moiety as Antiviral Agents.

A series of vanillin derivatives incorporating a bis(2-hydroxyethyl)dithioacetal moiety was designed and synthesized via a facile method. A plausible reaction pathway was proposed and verified by computational studies. Bioassay results demonstrated that target compounds possessed good to excellent activities against potato virus Y (PVY) and cucumber mosaic virus (CMV), of which, compound 6f incorporating a bis(2-hydroxyethyl)dithioacetal moiety, exhibited the best curative and protection activities against PVY and CMV in vivo, with 50% effective concentration values of 217.6, 205.7 µg/mL and 206.3, 186.2 µg/mL, respectively, better than those of ribavirin (848.0, 808.1 µg/mL and 858.2, 766.5 µg/mL, respectively), dufulin (462.6, 454.8 µg/mL and 471.2, 465.4 µg/mL, respectively), and ningnanmycin (440.5, 425.3 µg/mL and 426.1, 405.3 µg/mL, respectively). Current studies provide support for the application of vanillin derivatives incorporating bis(2-hydroxyethyl)dithioacetal as new antiviral agents.

Design, synthesis, antiviral bioactivity and three-dimensional quantitative structure-activity relationship study of novel ferulic acid ester derivatives containing quinazoline moiety.

BACKGROUND: Ferulic acid and quinazoline derivatives possess good antiviral activities. In order to develop novel compounds with high antiviral activities, a series of ferulic acid ester derivatives containing quinazoline were synthesized and evaluated for their antiviral activities. RESULTS: Bioassays indicated that some of the compounds exhibited good antiviral activities in vivo against tobacco mosaic virus (TMV) and cucumber mosaic virus (CMV). One of the compounds demonstrated significant curative and protective activities against TMV and CMV, with EC50 values of 162.14, 114.61 and 255.49, 138.81 mg L-1 , respectively, better than those of ningnanmycin (324.51, 168.84 and 373.88, 272.70 mg L-1 ). The values of q2 and r2 for comparative molecular field analysis and comparative molecular similarity index analysis in the TMV (0.508, 0.663 and 0.992, 0.930) and CMV (0.530, 0.626 and 0.997, 0.981) models presented good predictive abilities. CONCLUSION: Some of the title compounds demonstrated good antiviral activities. Three-dimensional quantitative structure-activity relationship models revealed that the antiviral activities depend on steric and electrostatic properties. These results could provide significant structural insights for the design of highly active ferulic acid derivatives. © 2017 Society of Chemical Industry.

Design, synthesis, and antiviral activities of 1,5-benzothiazepine derivatives containing pyridine moiety.

In our previous work, a series of novel benzothiazepine derivatives containing pyridine moiety were successfully synthesized through chalcone 1,3-dipolar cycloaddition and determined their antiviral activity against tobacco mosaic virus (TMV). Bioassay results indicated that most of these target compounds exhibited improved curative, protection, and inactivation activity in vivo than the commercial agent ningnanmycin. Particularly, compound 3m exhibited marked curative activity against TMV, with an EC50 value of 352.2 µM, which was even better than that of ningnanmycin. The compound was identified as the most promising candidate for inhibiting plant virus and an excellent compound with antiviral activities against TMV. Structure-activity relationship experiment indicated that the 1,5-benzothiazepine moiety is crucial for potent anti-TMV activity.

Antibacterial Activity and Mechanism of Action of Sulfone Derivatives Containing 1,3,4-Oxadiazole Moieties on Rice Bacterial Leaf Blight.

In this study, sulfone derivatives containing 1,3,4-oxadiazole moieties indicated good antibacterial activities against rice bacterial leaf blight caused by the pathogen Xanthomonas oryzaepv. pv. oryzae (Xoo). In particular, 2-(methylsulfonyl)-5-(4-fluorobenzyl)-1,3,4-oxadiazole revealed the best antibacterial activity against Xoo, with a half-maximal effective concentration (EC50) of 9.89 µg/mL, which was better than those of the commercial agents of bismerthiazole (92.61 µg/mL) and thiodiazole copper (121.82 µg/mL). In vivo antibacterial activity tests under greenhouse conditions and field trials demonstrated that 2-(methylsulfonyl)-5-(4-fluorophenyl)-1,3,4-oxadiazole was effective in reducing rice bacterial leaf blight. Meanwhile, 2-(methylsulfonyl)-5-(4-fluorophenyl)-1,3,4-oxadiazole stimulate the increase in superoxide dismutase (SOD) and peroxidase (POD) activities in rice, causing marked enhancement of plant resistance against rice bacterial leaf blight. It could also improve the chlorophyll content and restrain the increase in the malondialdehyde (MDA) content in rice to considerably reduce the amount of damage caused by Xoo. Moreover, 2-(methylsulfonyl)-5-(4-fluorophenyl)-1,3,4-oxadiazole, at a concentration of 20 µg/mL, could inhibit the production of extracellular polysaccharide (EPS) with an inhibition ratio of 94.52%, and reduce the gene expression levels of gumB, gumG, gumM, and xanA, with inhibition ratios of 94.88%, 68.14%, 86.76%, and 79.21%, respectively.