Identification of two major QTLs for pod shell thickness in peanut (Arachis hypogaea L.) using BSA-seq analysis.

BACKGROUND: Pod shell thickness (PST) is an important agronomic trait of peanut because it affects the ability of shells to resist pest infestations and pathogen attacks, while also influencing the peanut shelling process. However, very few studies have explored the genetic basis of PST. RESULTS: An F2 segregating population derived from a cross between the thick-shelled cultivar Yueyou 18 (YY18) and the thin-shelled cultivar Weihua 8 (WH8) was used to identify the quantitative trait loci (QTLs) for PST. On the basis of a bulked segregant analysis sequencing (BSA-seq), four QTLs were preliminarily mapped to chromosomes 3, 8, 13, and 18. Using the genome resequencing data of YY18 and WH8, 22 kompetitive allele-specific PCR (KASP) markers were designed for the genotyping of the F2 population. Two major QTLs (qPSTA08 and qPSTA18) were identified and finely mapped, with qPSTA08 detected on chromosome 8 (0.69-Mb physical genomic region) and qPSTA18 detected on chromosome 18 (0.15-Mb physical genomic region). Moreover, qPSTA08 and qPSTA18 explained 31.1-32.3% and 16.7-16.8% of the phenotypic variation, respectively. Fifteen genes were detected in the two candidate regions, including three genes with nonsynonymous mutations in the exon region. Two molecular markers (Tif2_A08_31713024 and Tif2_A18_7198124) that were developed for the two major QTL regions effectively distinguished between thick-shelled and thin-shelled materials. Subsequently, the two markers were validated in four F2:3 lines selected. CONCLUSIONS: The QTLs identified and molecular markers developed in this study may lay the foundation for breeding cultivars with a shell thickness suitable for mechanized peanut shelling.

Effects of Neutron and Gamma Rays Combined Irradiation on the Transcriptional Profile of Human Peripheral Blood.

We studied the effects of neutrons, neutrons and γ rays, and γ rays exposures on the transcription spectrum in human peripheral blood of three healthy adult men. Samples were irradiated with 1.42 Gy 2.5-MeV neutrons, 0.71 Gy neutrons and 0.71 Gy 137Cs γ rays, and 1.42 Gy 137Cs γ rays. Transcriptome sequencing identified 56 differentially co-expressed genes and enriched 26 KEGG pathways. There are 97, 45 and 30 differentially expressed genes in neutron, neutron and γ ray combined treatment, and γ rays, respectively, and 21, 3 and 8 KEGG pathways with significant differences are enriched. Fluorescence quantitative polymerase chain reaction (qPCR) verified differential co-expression of AEN, BAX, DDB2, FDXR, and MDM2. Additionally, irradiation of AHH-1 human lymphocytes with a 252Cf neutron source at 0, 0.14, 0.35, and 0.71 Gy, fluorescence qPCR revealed a dose-response relationship for BAX, DDB2, and FDXR at dose ranges of 0-0.71 Gy, with R2 of 0.803, 0.999, and 0.999, respectively. Thus, neutrons can induce more differentially expressed genes and enrich more pathways. Combined treatment of neutrons and γ-rays may incorporate damage of both high and low LET, the genes activated by neutrons and γ rays combined are almost the combination of genes activated by neutron and γ rays combined treatment. BAX, DDB2 and FDXR are differentially expressed after irradiation by Deuterium-Deuterium (D-D) neutron source and 252Cf neutron source, so they are expected to be molecular targets of neutron damage.

Gut microbiota of two invasive fishes respond differently to temperature.

Temperature variation structures the composition and diversity of gut microbiomes in ectothermic animals, key regulators of host physiology, with potential benefit to host or lead to converse results (i.e., negative). So, the significance of either effect may largely depend on the length of time exposed to extreme temperatures and how rapidly the gut microbiota can be altered by change in temperature. However, the temporal effects of temperature on gut microbiota have rarely been clarified. To understand this issue, we exposed two juvenile fishes (Cyprinus carpio and Micropterus salmoides), which both ranked among the 100 worst invasive alien species in the world, to increased environmental temperature and sampled of the gut microbiota at multiple time points after exposure so as to determine when differences in these communities become detectable. Further, how temperature affects the composition and function of microbiota was examined by comparing predicted metagenomic profiles of gut microbiota between treatment groups at the final time point of the experiment. The gut microbiota of C. carpio was more plastic than those of M. salmoides. Specifically, communities of C. carpio were greatly altered by increased temperature within 1 week, while communities of M. salmoides exhibit no significant changes. Further, we identified 10 predicted bacterial functional pathways in C. carpio that were temperature-dependent, while none functional pathways in M. salmoides was found to be temperature-dependent. Thus, the gut microbiota of C. carpio was more sensitive to temperature changes and their functional pathways were significantly changed after temperature treatment. These results showed the gut microbiota of the two invasive fishes differ in response to temperature change, which may indicate that they differ in colonization modes. Broadly, we have confirmed that the increased short-term fluctuations in temperatures are always expected to alter the gut microbiota of ectothermic vertebrates when facing global climate change.

Research on the Relationship between the Amylopectin Structure and the Physicochemical Properties of Starch Extracted from Glutinous Rice.

Glutinous rice has very low amylose content and is a good material for determining the structure and physicochemical properties of amylopectin. We selected 29 glutinous rice varieties and determined the amylopectin structure by high-performance anion exchange chromatography with the pulsed amperometric detection method. We also determined the correlation between amylopectin structure and the physicochemical properties of starch extracted from these varieties. The results showed that the amylopectin chain ratio Σdegree of polymerization (DP) ≤ 11/ΣDP ≤ 24 of 29 glutinous rice varieties was greater than 0.26, signifying that these varieties contained type II amylopectin. The results of the correlation analysis with gelatinization temperature showed that ΣDP 6-11 was significantly negatively correlated with the onset gelatinization temperature (GT) (TO), peak GT (TP), and conclusion GT (TC). Among the thermodynamic properties, ΣDP 12-24 was significantly positively correlated with To, Tp, and Tc, ΣDP 25-36 was significantly negatively correlated with To, Tp, and Tc, and ΣDP ≥ 37 had no correlation with the thermodynamic properties. The results of correlation analysis with RVA spectrum characteristic values showed that ΣDP 6-11 was significantly negatively correlated with hot paste viscosity (HPV), cool paste viscosity (CPV), consistency viscosity (CSV), peak time (PeT), and pasting temperature (PaT) among the Rapid Visco Analyzer (RVA) profile characteristics, ΣDP 12-24 was significantly positively correlated with HPV, CPV, CSV, PeT, and PaT, and ΣDP ≥ 25 had no correlation with the viscosity characteristics. Therefore, we concluded that the amylopectin structure had a greater effect on the TO, TP, TC, ΔH and peak viscosity, HPV, CPV, CSV, PeT, and PaT. The glutinous rice varieties with a higher distribution of short chains and a lower distribution of medium and long chains in the amylopectin structure resulted in lower GT and RVA spectrum characteristic values.

Hydrogen sulfide inhibits human T-cell leukemia virus type-1 (HTLV-1) protein expression via regulation of ATG4B.

Hydrogen sulfide (H2 S) is a redox gasotransmitter. It has been shown that H2 S has a key role in host antiviral defense by inhibiting interleukin production and S-sulfhydrating Keap1 lead to Nrf2/ARE pathway activation. However, it is yet unclear whether H2 S can play an antiviral role by regulating autophagy. In this study, we found that exogenous H2 S decreased the expression of human T-cell leukemia virus type-1 (HTLV-1) protein and HTLV-1 induced autophagosomes accumulation. Transmission electron microscope assays indicated that autophagosomes accumulation decreased after H2 S administration. HTLV-1-transformed T-cell lines had a high level of CSE (H2 S endogenous enzyme) which could be induced in Hela by HTLV-1 infection. Immunoblot demonstrated that overexpression of CSE inhibited HTLV-1 protein expression and autophagy. And we got the opposite after CSE knockdown. Meanwhile, H2 S could not restrain the autophagy when ATG4B had a mutant at its site of 89. In a word, these results suggested that H2 S modulated HTLV-1 protein expression via ATG4B. Therefore, our findings suggested a new mechanism by which H2 S defended against virus infection.

ESTABLISHMENT OF THE IN VITRO DOSE-RESPONSE CALIBRATION CURVE FOR X-RAY-INDUCED MICRONUCLEI IN HUMAN LYMPHOCYTES.

The cytokinesis-block micronucleus assay has proven to be a reliable technique for biological dosimetry. This study aimed to establish the dose-response curve for X-ray-induced micronucleus. Peripheral blood samples from three healthy donors were irradiated with various doses and scoring criteria by the micronuclei (MN) in binucleated cells. The results showed that the frequency of MN increased with the elevation of radiation dose. CABAS and Dose Estimate software were used to fit the MN and dose into a linear quadratic model, and the results were compared. The linear and quadratic coefficients obtained by the two software were basically the same and were comparable with published curves of similar radiation quality and dose rates by other studies. The dose-response curve established in this study can be used as an alternative method for in vitro dose reconstruction and provides a reliable tool for biological dosimetry in accidental or occupational radiation exposures.

Implementing an Air Suction Effect Induction Strategy to Create Super Thermally Insulating and Superelastic SiC Aerogels.

Silicon carbide (SiC) aerogels are promising thermal insulators that are lightweight and possess high thermal stability. However, their application is hindered by their brittleness. Herein, an air suction effect induction (ASEI) strategy is proposed to fabricate a super thermally insulating SiC aerogel (STISA). The ASEI strategy exploits the air suction effect to subtly regulate the directional flow of the SiO gas, which can induce directional growth and assembly of SiC nanowires to form a directional lamellar structure. The sintering time is significantly reduced by >90%. Significant improvements in the compression and elasticity performance of the STISA are achieved upon the formation of a directional lamellar structure through the ASEI strategy. Moreover, the lamellar structure endows the STISA with an ultralow thermal conductivity of 0.019 W m-1 K-1 . The ASEI strategy paves the way for structural design of advanced ceramic aerogels for super thermal insulation.

Transcriptome analysis of pod mutant reveals plant hormones are important regulators in controlling pod size in peanut (Arachis hypogaea L.).

Pod size is an important yield-influencing trait in peanuts. It is affected by plant hormones and identifying the genes related to these hormones may contribute to pod-related trait improvements in peanut breeding programs. However, there is limited information on the molecular mechanisms of plant hormones that regulate pod size in peanuts. We identified a mutant with an extremely small pod (spm) from Yuanza 9102 (WT) by 60Co γ-radiation mutagenesis. The length and width of the natural mature pod in spm were only 71.34% and 73.36% of those in WT, respectively. We performed comparative analyses for morphological characteristics, anatomy, physiology, and global transcriptome between spm and WT pods. Samples were collected at 10, 20, and 30 days after peg elongation into the soil, representing stages S1, S2, and S3, respectively. The differences in pod size between WT and spm were seen at stage S1 and became even more striking at stages S2 and S3. The cell sizes of the pods were significantly smaller in spm than in WT at stages S1, S2, and S3. These results suggested that reduced cell size may be one of the important contributors for the small pod in spm. The contents of indole-3-acetic acid (IAA), gibberellin (GA), and brassinosteroid (BR) were also significantly lower in spm pods than those in WT pods at all three stages. RNA-Seq analyses showed that 1,373, 8,053, and 3,358 differently expressed genes (DEGs) were identified at stages S1, S2, and S3, respectively. Functional analyses revealed that a set of DEGs was related to plant hormone biosynthesis, plant hormone signal transduction pathway, and cell wall biosynthesis and metabolism. Furthermore, several hub genes associated with plant hormone biosynthesis and signal transduction pathways were identified through weighted gene co-expression network analysis. Our results revealed that IAA, GA, and BR may be important regulators in controlling pod size by regulating cell size in peanuts. This study provides helpful information for the understanding of the complex mechanisms of plant hormones in controlling pod size by regulating the cell size in peanuts and will facilitate the improvement of peanut breeding.

High Performance and Self-Humidifying of Novel Cross-Linked and Nanocomposite Proton Exchange Membranes Based on Sulfonated Polysulfone.

The introduction of inorganic additive or nanoparticles into fluorine-free proton exchange membranes (PEMs) can improve proton conductivity and have considerable effects on the performance of polymer electrolyte membrane fuel cells. Based on the sol-gel method and in situ polycondensation, novel cross-linked PEM and nanocomposite PEMs based on a sulfonated polysulfone (SPSU) matrix were prepared by introducing graphene oxide (GO) polymeric brushes and incorporating Pt-TiO2 nanoparticles into an SPSU matrix, respectively. The results showed that the incorporation of Pt-TiO2 nanoparticles could obviously enhance self-humidifying and thermal stability. In addition, GO polymer brushes fixed on polymeric PEM by forming a cross-linked network structure could not only solve the leakage of inorganic additives during use and compatibility problem with organic polymers, but also significantly improve proton conductivity and reduce methanol permeability of the nanocomposite PEM. Proton conductivity, water uptake and methanol permeability of the nanocomposite PEM can be up to 6.93 mS cm-1, 46.58% and be as low as 1.4157 × 10-6 cm2 s-1, respectively, which represent increases of about 70%, about 22% and a decrease of about 40%, respectively, compared with that of primary SPSU. Therefore, the synergic action of the covalent cross-linking, GO polymer brush and nanoparticles can significantly and simultaneously improve the overall performance of the composite PEM.

Improvement of the Thermal Insulation Performance of Silica Aerogel by Proper Heat Treatment: Microporous Structures Changes and Pyrolysis Mechanism.

A simple heat treatment method was used to optimize the three-dimensional network structure of the hydrophobic aerogel, and during the heat treatment process at 200-1000 °C, the thermal conductivity of the aerogel reached the lowest to 0.02240 W/m·K between 250 °C and 300 °C, which was mainly due to the optimization of microstructure and pyrolysis of surface groups. Further Fluent heat-transfer simulation also confirmed the above results. Synchrotron vacuum ultraviolet photoionization mass spectrometry (SVUV-PIMS) was used to finely measure the pyrolysis process of aerogels, and the pyrolysis process of aerogel was divided into four stages. (I) Until 419 °C, as the temperature continued to rise, surface methyl groups were oxidized to form hydroxyl. (II) As the temperature reached to 232 °C, the oxidation proceeded. In addition, inside the aerogel, because of lacking oxygen, the reaction produced CH4 and C-Si bonds would form. (III) After 283 °C, Si-OH groups began to condense to form Si-O-Si, which optimized the three-dimensional network structures to be beneficial to improve the thermal insulation performance of silica aerogel. (IV) When it reached 547 °C, the chemical reaction was terminated, and all the primary particles gradually fused into secondary particles and sintered to form clusters.

AhNPR3 regulates the expression of WRKY and PR genes, and mediates the immune response of the peanut (Arachis hypogaea L.).

Systemic acquired resistance is an essential immune response that triggers a broad-spectrum disease resistance throughout the plant. In the present study, we identified a peanut lesion mimic mutant m14 derived from an ethyl methane sulfonate-mutagenized mutant pool of peanut cultivar "Yuanza9102." Brown lesions were observed in the leaves of an m14 mutant from seedling stage to maturity. Using MutMap together with bulked segregation RNA analysis approaches, a G-to-A point mutation was identified in the exon region of candidate gene Arahy.R60CUW, which is the homolog of AtNPR3 (Nonexpresser of PR genes) in Arabidopsis. This point mutation caused a transition from Gly to Arg within the C-terminal transactivation domain of AhNPR3A. The mutation of AhNPR3A showed no effect in the induction of PR genes when treated with salicylic acid. Instead, the mutation resulted in upregulation of WRKY genes and several PR genes, including pathogenesis-related thaumatin- and chitinase-encoding genes, which is consistent with the resistant phenotype of m14 to leaf spot disease. Further study on the AhNPR3A gene will provide valuable insights into understanding the molecular mechanism of systemic acquired resistance in peanut. Moreover, our results indicated that a combination of MutMap and bulked segregation RNA analysis is an effective method for identifying genes from peanut mutants.

Effects of Cellulase and Lactobacillus plantarum on Fermentation Quality, Chemical Composition, and Microbial Community of Mixed Silage of Whole-Plant Corn and Peanut Vines.

Significant anaerobic fermentation occurs in silage through the action of anaerobic bacteria. The objective of this study was to evaluate the effects of cellulase and Lactobacillus plantarum on the fermentation quality and bacterial community of whole-plant corn and peanut vine mixed silage. Mixed silage was tested with no addition (CK), addition of Lactobacillus plantarum (LP), addition of cellulase (CE), and the simultaneous addition of Lactobacillus plantarum and cellulase (LPCE). LPCE samples exhibited decreased pH; decreased content of acetic acid, propionic acid, and butyric acid; and increased content of lactic acid. LP and LPCE had better effects on chemical composition than CK and CE, especially in decreasing acid detergent fiber and neutral detergent fiber content. High-throughput sequencing identified Lactobacillus, Klebsiella, Serratia, and Weissella as the main microorganisms. LP and CE increased the abundance of Acetobacter, and LPCE decreased the abundance of Acetobacter. All additives decreased the abundance of Weissella, Leuconostoc, and Lactococcus, and increased the abundance of Pantoea. Overall, simultaneous addition of cellulase and Lactobacillus plantarum helped to improve the quality of mixed silage of whole-plant corn and peanut vines.

Mechanically Strong and Tailorable Polyimide Aerogels Prepared with Novel Silicone Polymer Crosslinkers.

Polyimide (PI) aerogels were prepared using self-designed silicone polymer cross-linkers with multi-amino from low-cost silane coupling agents to replace conventional small-molecule cross-linkers. The long-chain structure of silicone polymers provides more crosslinking points than small-molecule cross-linkers, thus improving the mechanical properties of polyimide. To investigate the effects of amino content and degree of polymerization on the properties of silicone polymers, the different silicone polymers and their cross-linked PI aerogels were prepared. The obtained PI aerogels exhibit densities as low as 0.106 g/cm3 and specific surface areas as high as 314 m2/g, and the maximum Young's modulus of aerogel is up to 20.9 MPa when using (T-20) as cross-linkers. The cross-linkers were an alternative to expensive small molecule cross-linkers, which can improve the mechanical properties and reduce the cost of PI aerogels.

Peripheral leukemia burden at time of apheresis negatively affects the clinical efficacy of CART19 in refractory or relapsed B-ALL.

Our previous clinical study achieved complete remission (CR) rates of >90% following chimeric antigen receptor T cells targeting CD19 (CART19) treatment of refractory/relapsed B cell acute lymphoblastic leukemia (r/r B-ALL); however, the influence of the leukemia burden in peripheral blood (PB) blasts remains unclear. Here, we retrospectively analyzed 143 patients treated with CART19 (including 36 patients with PB blasts) to evaluate the effect of peripheral leukemia burden at the time of apheresis. One hundred seventeen patients with high disease burdens achieved 91.5% CR or incomplete count recovery CR and 86.3% minimal residual disease-negative CR, and 26 patients with low disease burdens obtained 96.2% MRD- CR. Collectively, 9 of 36 (25%) patients with PB blasts and 2 of 107 (1.87%) patients without PB blasts did not respond to CART19 therapy. The leukemia burden in PB negatively influenced ex vivo cell characteristics, including the transduction efficiency of CD3+ T cells and their fold expansion, and in vivo cell dynamics, including peak CART19 proportion and absolute count, fold expansion, and persistence duration. Further studies showed that these patients had higher programmed death-1 expression in CART19 products. Our data imply that PB blasts negatively affected CART19 production and the clinical efficacy of CART19 therapy in patients with r/r B-ALL.

Quantitative Proteomics Analysis of Differentially Expressed Proteins in Serum of Former Uranium Miners by Isobaric Tags for the Relative and Absolute Quantitation.

The carcinogenicity of radon has been convincingly documented through epidemiological studies of underground miners. However, there is a lack of early warning indicators for radon radiation damage. In this study, mixed serum samples of 3 groups were collected from 27 underground uranium miners and seven aboveground miners according to the radiation exposure dose. The differentially expressed proteins in the serum were identified using the isobaric tags for the relative and absolute quantitation (iTRAQ)-based method. Some differentially expressed proteins were validated by enzyme-linked immunosorbent assay (ELISA) in 84 underground and 32 aboveground miners. A total of 25 co-differentially expressed proteins in 2 underground miner groups were screened, of which 9 were downregulated and 13 were upregulated. Biological process analysis of these proteins using Metascape showed that 5 GO terms were enriched, such as negative regulation of very-low-density lipoprotein particle clearance, endocytosis, and regulated exocytosis. The results of the ELISA for the expression levels of GCN1, CIP2A, and IGHV1-24 in the serum of 116 miners' serum showed that the levels of GCN1 and CIP2A were consistent with the iTRAQ results. In conclusion, APOC1, APOC2, APOC3, ORM1, ORM2, ANTXR1, GCN1, and CIP2A may be potential early markers of radon radiation damage.

"Robust-Soft" Anisotropic Nanofibrillated Cellulose Aerogels with Superior Mechanical, Flame-Retardant, and Thermal Insulating Properties.

Advanced thermal insulation materials with low thermal conductivity and robustness derived from regenerative resources are badly needed for building energy conservation. Among them, nanofibrillated cellulose aerogels have huge application potential in the field of thermal insulation materials, but it is still a challenge to prepare cellulose aerogels of excellent comprehensive properties in a simple way. Herein, we demonstrate a unidirectional freeze-drying strategy to develop a novel "robust-soft" anisotropic nanofibrillated cellulose aerogel (NFC-Si-T) by integrating nanofibrillated cellulose (NFC) and Si-O-Si bonding networks under the catalytic dehydration of p-toluenesulfonic acid (TsOH). The anisotropic structure endows the NFC-Si-T with high flexibility that can be easily bent or even tied with a knot, and in addition, it possesses high Young's modulus (1-3.66 MPa) that can resist the compression weight of 10,000 times of its own weight without deformation. Furthermore, the NFC-Si-T aerogels exhibit anisotropic thermal insulation performances with a low average thermal conductivity (0.028-0.049 W m-1 K-1). More importantly, the limited oxygen index of the NFC-Si-T reaches up to 42.6-51%, showing excellent flame-retardant performance. Therefore, the "robust-soft" anisotropic NFC-Si-T aerogels can be used as an advanced thermal insulation material for building thermal insulation applications.

Identification of the first Alu-mediated gross deletion involving the BCKDHA gene in a compound heterozygous patient with maple syrup urine disease.

AIMS: To investigate a family with clinical symptoms of maple syrup urine disease and reveal a genetic cause underlying this disease. METHODS: Targeted capture sequencing was used to screen for mutations in the patient. Real-Time PCR was carried out to perform exon 1, 5, 9 CNV analysis of samples from the patient's father, mother and sister. Whole genome sequencing was performed to map the approximate location of the break points of the gross deletion. Long-range PCR and Sanger sequencing were performed to identify the length of the deletion and to locate the break points. RESULTS: The patient is a compound heterozygous mutation including a small deletion mutation (c.1227_1229del chr19: 41930402) and a gross novel deletion including exon1-9 in BCKDHA. The junction site of the gross deletion was localized within a microhomologous sequence in two Alu elements. CONCLUSIONS: This study is the first time report on rearrangement sequences in BCKDHA mediated by Alu element, which resulted in MSUD. Our results may also offer new insights into the formation and pathogenicity of MSUD, and may be useful to genetic counseling and genetic testing.

Mechanically strong polyimide aerogels cross-linked with low-cost polymers.

Polyimide aerogels were prepared using low-cost polymers with different structure capped polyamide oligomers serving as cross-linking agents. To investigate the effects of the anhydride density on cross-linker chain units and side groups of cross-linkers on their properties and microstructures, two kinds of polymers from maleic anhydride, endic anhydride, and styrene were prepared by simple radical polymerization. The polyimide aerogels exhibit densities as low as 0.087 g cm-3 and specific surface areas as high as 456 m2 g-1. And the maximum modulus of the aerogel is up to 21.3 MPa. These cross-linkers are alternatives to expensive small molecule cross-linkers, therefore reducing the cost of PI aerogels.

Driver Fatigue Detection Based on Convolutional Neural Networks Using EM-CNN.

With a focus on fatigue driving detection research, a fully automated driver fatigue status detection algorithm using driving images is proposed. In the proposed algorithm, the multitask cascaded convolutional network (MTCNN) architecture is employed in face detection and feature point location, and the region of interest (ROI) is extracted using feature points. A convolutional neural network, named EM-CNN, is proposed to detect the states of the eyes and mouth from the ROI images. The percentage of eyelid closure over the pupil over time (PERCLOS) and mouth opening degree (POM) are two parameters used for fatigue detection. Experimental results demonstrate that the proposed EM-CNN can efficiently detect driver fatigue status using driving images. The proposed algorithm EM-CNN outperforms other CNN-based methods, i.e., AlexNet, VGG-16, GoogLeNet, and ResNet50, showing accuracy and sensitivity rates of 93.623% and 93.643%, respectively.