Effect of short implant crown-to-implant ratio on stress distribution in anisotropic bone with different osseointegration rates.

OBJECTIVE: This study aimed to provide evidence for the clinical application of single short implants by establishing an anisotropic, three-dimensional (3D) finite element mandible model and simulating the effect of crown-to-implant ratio (CIR) on biomechanics around short implants with different osseointegration rates. METHODS: Assuming that the bone is transversely isotropic by finite element method, we created four distinct models of implants for the mandibular first molar. Subsequently, axial and oblique forces were applied to the occlusal surface of these models. Ultimately, the Abaqus 2020 software was employed to compute various mechanical parameters, including the maximum von Mises stress, tensile stress, compressive stress, shear stress, displacement, and strains in the peri-implant bone tissue. RESULTS: Upon establishing consistent osseointegration rates, the distribution of stress exhibited similarities across models with varying CIRs when subjected to vertical loads. However, when exposed to inclined loads, the maximum von Mises stress within the cortical bone escalated as the CIR heightened. Among both loading scenarios, notable escalation in the maximum von Mises stress occurred in the model featuring a CIR of 2.5 and an osseointegration rate of 25%. Conversely, other models displayed comparable strength. Notably, stress and strain values uniformly increased with augmented osseointegration across all models. Furthermore, an increase in osseointegration rate correlated with reduced maximum displacement for both cortical bone and implants. CONCLUSIONS: After fixing osseointegration rates, the stress around shorter implants increased as the CIR increased under inclined loads. Thus, the effect of lateral forces should be considered when selecting shorter implants. Moreover, an implant failure risk was present in cases with a CIR ≥ 2.5 and low osseointegration rates. Additionally, the higher the osseointegration rate, the more readily the implant can achieve robust stability.

Three-dimensional finite element analysis of stress distribution on short implants with different bone conditions and osseointegration rates.

OBJECTIVE: This experiment aimed to investigate the effects of bone conditions and osseointegration rates on the stress distribution of short implants using finite element analysis and also to provide some reference for the application of short implants from a biomechanical prospect. MATERIALS AND METHODS: Anisotropic jaw bone models with three bone conditions and 4.1 × 6 mm implant models were created, and four osseointegration rates were simulated. Stress and strain for the implants and jaws were calculated during vertical or oblique loading. RESULTS: The cortical bone area around the implant neck was most stressed. The maximum von Mises stress in cortical bone increased with bone deterioration and osseointegration rate, with maximum values of 144.32 MPa and 203.94 MPa for vertical and inclined loading, respectively. The osseointegration rate had the greatest effect on the maximum principal stress in cortical bone of type III bone, with its value increasing by 63.8% at a 100% osseointegration rate versus a 25% osseointegration rate. The maximum and minimum principal stresses under inclined load are 1.3 ~ 1.7 and 1.4 ~ 1.8 times, respectively, those under vertical load. The stress on the jaw bone did not exceed the threshold when the osseointegration rate was ≥ 50% for Type II and 100% for Type III. High strain zones are found in cancellous bone, and the maximum strain increases as the bone condition deteriorate and the rate of osseointegration decreases. CONCLUSIONS: The maximum stress in the jaw bone increases as the bone condition deteriorates and the osseointegration rate increases. Increased osseointegration rate reduces cancellous bone strain and improves implant stability without exceeding the yield strength of the cortical bone. When the bone condition is good, and the osseointegration ratio is relatively high, 6 mm short implants can be used. In clinical practice, incline loading is an unfavorable loading condition, and axial loading should be used as much as possible.

ß-Cyclodextrin and folic acid host-guest interaction binding parameters determined by Taylor dispersion analysis and affinity capillary electrophoresis.

The thermodynamic properties of molecular recognition in host-guest inclusion complexes can be studied by Taylor dispersion analysis (TDA). Host-guest inclusion complexes have modest size, and it is possible to get convergent results fast, achieving greater certainty for the obtained thermodynamic properties. Cyclodextrins (CDs) and their derivatives can be used as drug carriers that can boost stability, solubility, and bioavailability of physiologically active substances. A simple and effective approach for assessing the binding properties of CD complexes that are critical in the early stages of drug and formulation development is needed to fully understand the process of CD and guest molecules' complex formation. In this work, TDA was successfully used to rapidly determine interaction parameters, including binding constant and stoichiometry, between ß-CD and folic acid (FA) along with the diffusivities of the free FA and its complex with ß-CD. Additionally, the FA diffusion coefficient obtained by TDA was compared to the results previously obtained by nuclear magnetic resonance. Affinity capillary electrophoresis (ACE) was also used to compare the binding constants obtained by different methods. The results showed that the binding constants obtained by ACE were somewhat lower than those obtained by the two TDA procedures.

Fungistatic Mechanism of Ammonia against Nematode-Trapping Fungus Arthrobotrys oligospora, and Strategy for This Fungus To Survive Ammonia.

Soil fungistasis is a phenomenon in which the germination and growth of fungal propagules is widely inhibited in soils. Although fungistatic compounds are known to play important roles in the formation of soil fungistasis, how such compounds act on soil fungi is little studied. In this study, it was found that ammonia (NH3) induced global protein misfolding marked by increased ubiquitination levels of proteins (ubiquitylome data and Western blot verification). The misfolded proteins should trigger the endoplasmic reticulum (ER) stress, which was indicated by electron microscope image and proteome data. Results from the mutants of BiP and proteasome subunit alpha 7 suggested that ER stress played a mechanistic role in inhibiting conidial germination. Results from proteome data indicated that, to survive ammonia fungistasis, conidia first activated the unfolded protein response (UPR) to decrease ER stress and restore ER protein homeostasis, and the function of UPR in surviving ammonia was confirmed by using mutant strains. Second, ammonia toxicity could be reduced by upregulating carbon metabolism-related proteins, which benefited ammonia fixation. The results that metabolites (especially glutamate) could relieve the ammonia fungistasis confirmed this indirectly. Finally, results from gene knockout mutants also suggested that the fungistatic mechanism of ammonia is common for soil fungistasis. This study increased our knowledge regarding the mechanism of soil fungistasis and provided potential new strategies for manipulating soil fungistasis. IMPORTANCE Soil fungistasis is a phenomenon in which the germination and growth of fungal propagules is widely inhibited in soil. Although fungistatic compounds are known to play important roles in the formation of soil fungistasis, how such compounds act on soil fungi remains little studied. This study revealed an endoplasmic reticulum stress-related fungistatic mechanism with which ammonia acts on Arthrobotrys oligospora and a survival strategy of conidia under ammonia inhibition. Our study provides the first mechanistic explanation of how ammonia impacts fungal spore germination, and the mechanism may be common for soil fungistasis. This study increases our knowledge regarding the mechanism of soil fungistasis in fungal spores and provides potential new strategies for manipulating soil fungistasis.

Detecting the formation of human c-KIT oncogene promoter G-Quadruplex by Taylor dispersion analysis.

The formation of G-quadruplex (G4) structures in oncogenic G-rich promoter regions are implicated in their biological functions, especially the inhibition of transcription. The binding of cations is thought to contribute to the stabilization of the G4 formation and competition against the duplex formation in the genomic sequence. Furthermore, it might affect the recognition of DNA-binding proteins. Therefore, measuring the interaction between G4 DNA and cations in a free solution environment is critical for evaluating G4 DNA biological functions. However, how binding to cations (K+ and NH4+) affects the folding equilibrium of the G4 structure remains unclear. In this work, a Taylor dispersion analysis (TDA) method using a capillary electrophoresis (CE) instrument was established for the quantitative characterization of the cation-dependent G4 formation in the human c-KIT oncogene promoter region, as well as diffusivities and hydrodynamic radii of DNA variations before and after folding. Our results showed that both K+ and NH4+ can induce the random-coiled c-KIT DNA to unfold and form a more unstretched intermediate state and then fold into tightly structured G4s with smaller size. The G4 size induced by NH4+ was smaller than that induced by K+ ions, though these two cations induced the c-KIT G4 DNA formation with similar binding constants (order of magnitude around 106 M-1). The TDA method can be widely used for rapid structural analyses of trace amounts of DNA mixtures, which effectively differentiate DNA variations or DNA-ligand complex conformations.

Remediation of Cd-Contaminated Soil by Modified Nanoscale Zero-Valent Iron: Role of Plant Root Exudates and Inner Mechanisms.

In this study, the role of exogenous root exudates and microorganisms was investigated in the application of modified nanoscale zero-valent iron (nZVI) for the remediation of cadmium (Cd)-contaminated soil. In this experiment, citric acid (CA) was used to simulate root exudates, which were then added to water and soil to simulate the pore water and rhizosphere environment. In detail, the experiment in water demonstrated that low concentration of CA facilitated Cd removal by nZVI, while the high concentration achieved the opposite. Among them, CA can promote the adsorption of Cd not only by direct complexation with heavy metal ions, but also by indirect effect to promote the production of iron hydroxyl oxides which has excellent heavy metal adsorption properties. Additionally, the H+ dissociated from CA posed a great influence on Cd removal. The situation in soil was similar to that in water, where low concentrations of CA contributed to the immobilization of Cd by nZVI, while high concentrations promoted the desorption of Cd and the generation of CA-Cd complexes which facilitated the uptake of Cd by plants. As the reaction progressed, the soil pH and cation exchange capacity (CEC) increased, while organic matter (OM) decreased. Meanwhile, the soil microbial community structure and diversity were investigated by high-throughput sequencing after incubation with CA and nZVI. It was found that a high concentration of CA was not conducive to the growth of microorganisms, while CMC had the effect of alleviating the biological toxicity of nZVI.

Quantitative characterization of human oncogene promoter G-quadruplex DNA-ligand interactions using a combination of mass spectrometry and capillary electrophoresis.

Human c-KIT oncogene is known to regulate cell growth and proliferation, and thus, acts as a probable target in the treatment of gastrointestinal tumors (GIST). To identify small molecule ligands which can specifically bind with the G-quadruplex (G4) in the c-KIT promoter region as potential antitumor agents, we propose the combination of electrospray ionization-mass spectrometry (ESI-MS), capillary electrophoresis frontal analysis (CE-FA), and Taylor dispersion analysis (TDA) to accurately investigate the G4/ligands binding properties. First, ESI-MS was used for initial screening of natural products (NPs). CE-FA was then used to calculate specific binding constants and the stoichiometry of the native state binding pair in solution. Next, TDA, a micro-capillary flow technique was used to examine the effect of the ligand binding on the diffusivity and particle size of the c-KIT G4. Two of the screened NPs, scopolamine butylbromide (L1) and isorhamnetin-3-O-neohesperidoside (L3), were found to specifically bind to the c-KIT G4 with binding constants of around 104 M-1 and 1:1 stoichiometry in a free solution. TDA data showed that ligand binding (both L1 and L3) induced the c-KIT strands to fold into a tightly structured G4 with a decreased hydrodynamic radius. These ligands have the potential to be drug candidates for the regulation of c-KIT gene transcription by stabilizing the G4 structure. This methodology not only increased the speed of analysis but also improved its accuracy and specificity compared with the conventional binding approaches.

Methylglyoxal Has Different Impacts on the Fungistatic Roles of Ammonia and Benzaldehyde, and Lactoylglutathione Lyase Is Necessary for the Resistance of Arthrobotrys oligospora to Soil Fungistasis.

Biocontrol of root-knot nematode has attracted increasing attention over the past two decades. The inconsistent field performance of biocontrol agents, which is caused by soil fungistasis, often restricts their commercial application. There is still a lack of research on the genes involved in biocontrol fungi response to soil fungistasis, which is important for optimizing practical applications of biocontrol fungi. In this study, the lactoylglutathione lyase-encoding AOL_s00004g335 in the nematophagous fungi Arthrobotrys oligospora was knocked out, and three mutant strains were obtained. The hyphal growth of mutants on the three media was almost the same as that of the wild-type strain, but mutants had slightly higher resistance to NaCl, SDS, and H2O2. Methylglyoxal (MG) significantly increased the resistance of A. oligospora to ammonia, but decreased the resistance to benzaldehyde. Furthermore, the resistance of the mutants to soil fungistasis was largely weakened and MG could not increase the resistance of A. oligospora to soil fungistasis. Our results revealed that MG has different effects on the fungistatic roles of ammonia and benzaldehyde and that lactoylglutathione lyase is very important for A. oligospora to resist soil fungistasis.

Optical fiber sensor based on a cholesteric liquid crystal film for mixed VOC sensing.

This paper proposes a novel cholesteric liquid crystal (CLC) film-based dual-probe fiber sensor to monitor volatile organic compound (VOC) gas. The sensor consists of a 2×2 multimode fiber coupler, in which the two output fiber ends are covered by two types of CLC films (CLCF) with different pitches. It can be observed that the reflection peak wavelengths of CLCs shift to the red side as the VOC gas concentration increases. The sensitivities of the two CLCFs are 8.435 nm·L/mmol and 14.867 nm·L/mmol to acetone, 14.586 nm·L/mmol and 29.303 nm·L/mmol to ethanol, respectively. In addition, the dependence of the peak wavelength shift of CLCF on the total concentration of the acetone and ethanol mixed gas at different mixing ratios is measured. The linear relationships between the peak shift of CLCFs, the total mixed gas concentration and acetone/ethanol ratio are calculated using the least-squares method. Therefore, this proposed dual-probe fiber optic sensor can distinguish the concentrations of acetone and ethanol in a mixed gas of acetone and ethanol.

[Responses of the germinability of Sphagnum spores in peat to drainage in Baijianghe Peatland, China]. / 白江河泥炭地泥炭藓孢子萌发力对排水的响应.

Drainage severely changes the environment and ecological process in peatlands, but how does it affect the germinability of Sphagnum spores in peat remains unclear. In this study, we took two peat cores from a near-pristine stand dominated by Sphagnum and a drained stand dominated by dwarf shrubs in Baijianghe Peatland in the Changbai Mountains as experimental materials. Those peat cores were cut into slices. Physicochemical characteristics were measured while Sphagnum spores from each slice were extracted to count spore density and test spore germinability. After dating and determining relationship between peat depth and age, we tried to figure out the mechanism underlying the responses of Sphagnum spore germinability to drainage. The average number of spores in the near-pristine stand was slightly higher than that in the drained stand. There was no difference in average spore germinability between the two stands. The drained stand showed higher peat bulk density, total carbon and total nitrogen relative to the near-pristine stand. Upper peat core showed no significant difference in spore accumulation rate between the two stands after drainage (in 1987), with lower average spore germinability (34%) in the near-pristine stand relative to the drained stand (72%). For the whole peat cores, C/N was positively correlated with spore ger-minability in the near-pristine stand while total carbon, pH and burial time were negatively correlated with spore germinability in the drained stand. The drainage 30 years ago had limited effect on spore accumulation, but improved germinability of spores in shallow peat by changing physicochemical properties of peat due to accelerating decomposition, and thus reduced the persistence of spore bank. This may reduce the persistent regeneration potential of Sphagnum after catastrophic distur-bances.

[Determination of the binding of natural products to the human c-myb oncogene promoter G-quadruplex DNA by capillary electrophoresis and electrospray ionization mass spectrometry].

The relationship between a drug and its target directly affects its pharmacology and efficacy. Drug-target binding ability and binding stoichiometry are essential characterization data in pharmaceutical research. The c-myb proto-oncogene encodes a crucial transcription factor that is involved in proliferation, differentiation, and maturation during hematopoiesis. Recent studies have found that the human oncogene c-myb is overexpressed in cancer tissues such as colorectal cancer. C-myb has become a potential therapeutic target for colorectal cancer, leukemia, and other cancers. A guanine (G)-rich DNA sequence located in the promoter region of c-myb can be spontaneously folded to form an intra-molecular G-quadruplex (G4) with cationic induction. The specific recognition of small molecules can stabilize this G4 folding, thus regulating the transcription and expression of c-myb. In this study, pressure assisted capillary electrophoresis frontier analysis (PACE-FA) combined with electrospray ionization mass spectrometry (ESI-MS) was used to investigate the interactions between the human c-myb promoter G4 and natural product molecules. In PACE-FA, an external pressure (no more than 13.8 kPa) in the same direction of the migration of the analyte was used in capillary electrophoresis frontier analysis (CE-FA), which greatly sped up the analysis while maintaining the accuracy of the results. Meanwhile, the combination of PACE-FA and ESI-MS could rapidly determine the affinity and stoichiometric relationship between binding molecules and targets. First, the intramolecular parallel-stranded G4 formation of the c-myb promoter sequence in the presence of cations (K+, NH4+) was investigated by circular dichroism (CD) and ESI-MS. Then, ESI-MS was used to rapidly screen the natural products for candidate molecules that bound the target G4 DNA. The binding interactions were measured by mixing the c-myb DNA with each natural product separately in a 1:4 molar ratio, and then directly infusing these mixtures into the ESI-MS system. From the ESI-MS spectra, IRa values calculated from the relative intensities of DNA and its complex ions were used to probe the binding affinities of the natural products. This parameter denotes the relative binding affinity for a small molecule with the G4 DNA. Three natural products were identified through the screen, and their binding affinities to G4 DNA were ranked as follows:pseudolaric acid B > scopolamine butylbromide > nuciferine. Considering that both specific and non-specific binding existed in the solution phase, a free solution method using PACE-FA was developed to further test the binding ability of these products to the c-myb promoter G4 DNA. In the PACE-FA experiments, the pre-equilibrated mixture of the c-myb G4 DNA and the selected ligand was injected into the capillary prior to separation. Upon applying voltage and an external pressure (6.9 kPa) to the capillary, different species of analytes in the sample migrated at their own rates due to their different sizes and charges. The results showed that scopolamine butylbromide could bind specifically to target G4 DNA with 1:1 stoichiometry and a binding constant of 1.18×105 L/mol. Nuciferine's binding to G4 DNA showed a linear increasing trend due to nonspecific binding; thus nuciferine is a nonspecific binder. Although pseudolaric acid B showed high affinity for the c-myb G4 DNA and 1:1 and 1:2 G4-bound complex ions were observed in ESI-MS measurements, the PACE-FA results indicated that pseudolaric acid B did not bind to target G4 DNA in free solution. Therefore, scopolamine butylbromide could be the best candidate to regulate the transcription of the c-myb oncogene, and is expected to be a precursor for anticancer drugs. In this work, PACE-FA has allowed for significant improvements to the conventional CE-FA technique. This combination of PACE-FA and ESI-MS not only reduced the time needed for binding analysis, but also improved the accuracy and specificity of the affinity analysis compared to conventional binding approaches. Furthermore, this combination could be used to screen other targeted drug candidates and to evaluate their interaction mechanisms.

Biomaterials cross-linked graphene oxide composite aerogel with a macro-nanoporous network structure for efficient Cr (VI) removal.

Chitosan (CS) is an attractive bio-adsorbent in pollutant removal due to its environment-friendly properties and abundant adsorption sites. However, the weak mechanical properties and strong dissolubility in acidic conditions of CS hinder its wide application. Herein, a facile method was proposed to fabricate polydopamine (PDA) and CS cross-linked graphene oxide (GO) (GO/CS/PDA) composite aerogel for Cr (VI) removal. GO was cross-linked with CS, forming a reinforced and three-dimensional macroporous structure; the introduced PDA was simultaneously cross-linked with CS and GO, providing more abundant nanopores and active sites for Cr(VI) removal. Based on the batch experiment results, GO/CS/PDA exhibited an optimized mass ratio (1:20:2) of GO, CS, and PDA for the most effective Cr(VI) adsorption; the adsorption removal rate of Cr(VI) was pH dependent, with the highest removal rate at pH = 3.0. The pseudo-second-order kinetic and Freundlich models were more suitable for fitting the adsorption kinetics and adsorption isotherms, respectively, and the maximum adsorption capacity for GO/CS/PDA was 312.05 mg/g at 298 K. Thermodynamics parameters indicated that the adsorption was a spontaneous and exothermic process. The excellent mechanical integrity and reusable adsorption performance of GO/CS/PDA promise the adsorbent with satisfactory reusability.

[Simulated study on the effects of smoke on Sphagnum spore germination]. / çƒŸæ°”å¯¹æ³¥ç‚­è—“å­¢å­èŒå‘å½±å“çš„æ¨¡æ‹Ÿç ”ç©¶.

Moderate smoke could facilitate seed germination, but its effects on bryophyte spore germination is still unknown. Here, we analyzed the effects of smoke, capsule size and storage time on the spore germination of Sphagnum squarrosum and S. magellanicum, with the capsules of which being collected from two peatlands of the Changbai Mountains. The smoke solution prepared by burning peatland plants was combined with the capsules with different sizes (large, 2.10-2.50 mm in diameter; small, 1.50-1.90 mm in diameter) and storage time (old, being stored for 4.3 or 6.3 a; new, being stored for 0.3 a) to conduct a factorial experiment. The spores were soaked with smoke solution for different durations and then cultured for germination. The results showed that smoke solution affected spore germination. After 10 d cultivation, germination rate of spores soaking with smoke solution for all duration was increased by more than 5-fold, with the small spores having higher germination rate. After 21 d cultivation, the facilitative effect was only observed in moderate soaking (3 d), and spore size showed no effect on germination. Smoke solution could not increase the germination of spores from the capsules with long storage time (4.3 and 6.3 a). Our results indicated that moderate smoke solution soaking might accelerate germination of Sphagnum spores including small pores. In the ecosystems with casual fire disturbance such as peatlands, similar with its effects on the seed plants, smoke might play a key role in the regeneration and persistence of bryophyte population.

Comparative Transcriptomics Reveals Features and Possible Mechanisms of Glucose-Mediated Soil Fungistasis Relief in Arthrobotrys oligospora.

Soil-borne pest diseases result in large annual agricultural losses globally. Fungal bio-control agents are an alternative means of controlling pest diseases; however, soil fungistasis limits the effect of fungal agents. Nutrients can relieve soil fungistasis, but the mechanisms behind this process remain poorly understood. In this study, we determined and quantified the transcriptomes of Arthrobotrys oligospora, a nematode-trapping fungus, derived from samples of fresh conidia, germinated conidia, soil fungistatic conidia, and glucose-relieved conidia. The transcriptomes of fungistatic and glucose-relieved conidia were significantly different from those of the other two conidia samples. KEGG pathway analyses showed that those genes upregulated in fungistatic and glucose-relieved conidia were mainly involved in translation and substance metabolism, and the downregulated genes were mainly involved in MAPK pathway, autophagy, mitophagy, and endocytosis. As being different from the transcriptome of fungistatic conidia, upregulated genes in the transcriptome of glucose-relieved conidia are also related to replication and repair, spliceosome, oxidative phosphorylation, autophagy, and degradation pathway (lysosome, proteasome, and RNA degradation). And the upregulated genes resulted from comparison of glucose-relieved conidia and fungistatic conidia were enriched in metabolic pathways, cycle, DNA replication, and repair. The differentially splicing events in the transcriptome of glucose-relieved conidia are far more than that of other two transcriptomes, and genes regulated by differentially splicing were analyzed through KEGG pathway analysis. Furthermore, autophagy genes were proved to play important role in resisting soil fungistasis and glucose-mediated soil fungistasis relief. These data indicate that, in addition to being a carbon and energy source for conidia germination, glucose may also help to relieve soil fungistasis by activating many cellular processes, including autophagy, DNA replication and repair, RNA alternative splicing, and degradation pathways.

The lysine acetylome of the nematocidal bacterium Bacillus nematocida and impact of nematode on the acetylome.

Bacillus nematocida B16 (B16) is a pathogenic bacterium that is nematotoxic to plant-parasitic nematodes. In this study, we performed a quantitative lysine acetylome analysis on B16 to understand the potential roles of protein lysine acetylation on this host-pathogen interaction. Altogether, we identified 529 acetylation sites in 349 proteins, quantified 411 sites in 288 proteins, determined that the acetylation levels of 18 sites were up-regulated and those of 19 sites were down-regulated during pathogenesis. The acetylated proteins mainly participated in metabolic processes, protein synthesis, and cell wall/membrane biogenesis. Moreover, these proteins are involved in more than twenty KEGG pathways. Eight peptide motifs of acetylated proteins were identified, five of which have been thus far found only in the B16 acetylome. Twenty-two acetylated proteins were found to be involved in the synthesis of nematode attractants, and two were found to be involved in the secretion of virulence factors. In addition, the acetylation levels of ten lysine sites were regulated significantly differently in the presence of nematodes. Our results reveal that lysine acetylation may play roles in regulating B16-nematode interaction. SIGNIFICANCE: B. nematocida B16 is a bio-control bacterium against nematodes. It lures nematodes to their death by a Trojan horse mechanism. But there is little understanding about the regulation of this "Trojan horse" like pathogenesis. Lysine acetylation was reported to regulate diverse cellular processes. Our results revealed that lysine acetylation played indeed roles in regulating the B16-nematodes interaction. Our data laid a foundation for studying the molecule mechanism of lysine acetylation in regulating this host-pathogen interaction.