On the evolutionary trail of MagRs.

Magnetic sense, or termed magnetoreception, has evolved in a broad range of taxa within the animal kingdom to facilitate orientation and navigation. MagRs, highly conserved A-type iron-sulfur proteins, are widely distributed across all phyla and play essential roles in both magnetoreception and iron-sulfur cluster biogenesis. However, the evolutionary origins and functional diversification of MagRs from their prokaryotic ancestor remain unclear. In this study, MagR sequences from 131 species, ranging from bacteria to humans, were selected for analysis, with 23 representative sequences covering species from prokaryotes to Mollusca, Arthropoda, Osteichthyes, Reptilia, Aves, and mammals chosen for protein expression and purification. Biochemical studies revealed a gradual increase in total iron content in MagRs during evolution. Three types of MagRs were identified, each with distinct iron and/or iron-sulfur cluster binding capacity and protein stability, indicating continuous expansion of the functional roles of MagRs during speciation and evolution. This evolutionary biochemical study provides valuable insights into how evolution shapes the physical and chemical properties of biological molecules such as MagRs and how these properties influence the evolutionary trajectories of MagRs.

Polypeptide-PNP2 in Corn Cervi Pantotrichum Ameliorates Cognitive Impairment in Alzheimer's Disease Mice by Inhibiting Microglial Cell Activation.

We isolated a polypeptide PNP2 from Corn Cervi Pantotrichum and investigated its effect and mechanism on cognitive impairment in Alzheimer's disease (AD) mice. Morris water maze was used to assess the degree of cognitive impairment in mice. Histopathological changes were detected by H&E staining; the expressions of inflammatory cytokines were assayed by ELISA. Western blotting was employed to detect the protein expressions. PNP2 could improve cognitive impairment, central inflammatory response, and NLRP3 signaling in AD mice. In vitro experiments revealed that PNP2 could suppress the inflammatory response of microglial cells and reduce the activation of NLRP3 in microglial cells, while MCC950 could antagonize the effects of PNP2. Polypeptide component PNP2 in Corn Cervi Pantotrichum can ameliorate central nervous inflammation and cognitive impairment in AD mice by suppressing NLRP3 signaling.

Construction and evaluation of AuNPs enhanced electrochemical immunosensors with [Fe(CN)6]3-/4- and PPy probe for highly sensitive detection of human chorionic gonadotropin.

Human chorionic gonadotropin (HCG), a vital protein for pregnancy determination and a marker for trophoblastic diseases, finds application in monitoring early pregnancy and ectopic pregnancy. This study presents an innovative approach employing electrochemical immunosensors for enhanced HCG detection, utilizing Anti-HCG antibodies and gold nanoparticles (AuNPs) in the sensor platform. Two sensor configurations were optimized: BSA/Anti-HCG/c-AuNPs/MEL/e-AuNPs/SPCE with [Fe(CN)6]3-/4- as a redox probe (1) and BSA/Anti-HCG/PPy/e-AuNPs/SPCE using polypyrrole (PPy) as a redox probe (2). The first sensor offers linear correlation in the 0.10-500.00 pg∙mL-1 HCG range, with a limit of detection (LOD) of 0.06 pg∙mL-1, sensitivity of 32.25 µA∙pg-1∙mL∙cm-2, RSD <2.47 %, and a recovery rate of 101.03-104.81 %. The second sensor widens the HCG detection range (40.00 fg∙mL-1-5.00 pg∙mL-1) with a LOD of 16.53 fg∙mL-1, ensuring precision (RSD <1.04 %) and a recovery range of 94.61-106.07 % in serum samples. These electrochemical immunosensors have transformative potential in biomarker detection, offering enhanced sensitivity, selectivity, and stability for advanced healthcare diagnostics.

Highly Efficient and Stable Organic Solar Cells Enabled by a Commercialized Simple Thieno[3,2-b]thiophene Additive.

Delicately manipulating nanomorphology is recognized as a vital and effective approach to enhancing the performance and stability of organic solar cells (OSCs). However, the complete removal of solvent additives with high boiling points is typically necessary to maintain the operational stability of the device. In this study, two commercially available organic intermediates, namely thieno[3,2-b]thiophene (TT) and 3,6-dibromothieno[3,2-b]thiophene (TTB) are introduced, as solid additives in OSCs. The theoretical simulations and experimental results indicate that TT and TTB may exhibit stronger intermolecular interactions with the acceptor Y6 and donor PM6, respectively. This suggests that the solid additives (SAs) can selectively intercalate between Y6 and PM6 molecules, thereby improving the packing order and crystallinity. As a result, the TT-treated PM6:Y6 system exhibits a favorable morphology, improved charge carrier mobility, and minimal charge recombination loss. These characteristics contribute to an impressive efficiency of 17.75%. Furthermore, the system demonstrates exceptional thermal stability (T80 > 2800 h at 65 °C) and outstanding photostability. The universal applicability of TT treatment is confirmed in OSCs employing D18:L8-BO, achieving a significantly higher PCE of 18.3%. These findings underscore the importance of using appropriate solid additives to optimize the blend morphology of OSCs, thereby improving photovoltaic performance and thermal stability.

Pyrus betulaefolia ERF3 interacts with HsfC1a to coordinately regulate aquaporin PIP1;4 and NCED4 for drought tolerance.

Environmental disasters like drought reduce agricultural output and plant growth. Redox management significantly affects plant stress responses. An earlier study found that PbPIP1;4 transports H2O2 and promotes H2O2 downstream cascade signaling to restore redox equilibrium. However, this regulatory mechanism requires additional investigation. In this search, the AP2 domain-containing transcription factor was isolated by screening Y1H from the wild pear (Pyrus betulaefolia) cDNA library, named PbERF3. The overexpression of PbERF3 in pear callus and Arabidopsis enhanced plant resistance to drought and re-established redox balance. The transcripts of the NCEDs gene were upregulated under drought stress. The drought stress-related abscisic acid (ABA) signaling pathway modulates PbERF3. PbERF3 silencing lowered drought tolerance. Furthermore, yeast 2-hybrid, luciferase, bimolecular fluorescence complementation, and co-immunoprecipitation assays verified that PbERF3 physically interacted with PbHsfC1a. The PbERF3-PbHsfC1a heterodimer coordinately bound to PbPIP1;4 and PbNCED4 promoter, therefore activating both the H2O2 and the ABA signaling pathway. This work revealed a novel PbERF3-PbHsfC1a-PbNCED4-PbPIP1;4 regulatory module, in which PbERF3 interacts with PbHsfC1a to trigger the expression of target genes. This module establishes an interaction between the H2O2 signaling component PbPIP1;4 and the ABA pathways component PbNCED4, enabling a response to drought.

Multifunctional fluorescence/photoacoustic bimodal imaging of γ-glutamyltranspeptidase in liver disorders under different triggering conditions.

Hepatocellular carcinoma (HCC) seriously threatens the human health. Previous investigations revealed that γ-glutamyltranspeptidase (GGT) was tightly associated with the chronic injury, hepatic fibrosis, and the development of HCC, therefore might act as a potential indicator for monitoring the HCC-related processes. Herein, with the contribution of a structurally optimized probe ETYZE-GGT, the bimodal imaging in both far red fluorescence (FL) and photoacoustic (PA) modes has been achieved in multiple HCC-related models. To our knowledge, this work covered the most comprehensive models including the fibrosis and developed HCC processes as well as the premonitory induction stages (autoimmune hepatitis, drug-induced liver injury, non-alcoholic fatty liver disease). ETYZE-GGT exhibited steady and practical monitoring performances on reporting the HCC stages via visualizing the GGT dynamics. The two modes exhibited working consistency and complementarity with high spatial resolution, precise apparatus and desirable biocompatibility. In cooperation with the existing techniques including testing serum indexes and conducting pathological staining, ETYZE-GGT basically realized the universal application for the accurate pre-clinical diagnosis of as many HCC stages as possible. By deeply exploring the mechanically correlation between GGT and the HCC process, especially during the premonitory induction stages, we may further raise the efficacy for the early diagnosis and treatment of HCC.

Functional Differentiation of the Succinate Dehydrogenase Subunit SdhC Governs the Sensitivity to SDHI Fungicides, ROS Homeostasis, and Pathogenicity in Fusarium asiaticum.

Succinate dehydrogenase (SDH) is an integral component of the tricarboxylic acid cycle (TCA) and respiratory electron transport chain (ETC), targeted by succinate dehydrogenase inhibitors (SDHIs). Fusarium asiaticum is a prominent phytopathogen causing Fusarium head blight (FHB) on wheat. Here, we characterized the functions of the FaSdhA, FaSdhB, FaSdhC1, FaSdhC2, and FaSdhD subunits. Deletion of FaSdhA, FaSdhB, or FaSdhD resulted in significant growth defects in F. asiaticum. The FaSdhC1 or FaSdhC2 deletion mutants exhibited substantial reductions in fungal growth, conidiation, virulence, and reactive oxygen species (ROS). The FaSdhC1 expression was significantly induced by pydiflumetofen (PYD). The ΔFaSdhC1 mutant displayed hypersensitivity to SDHIs, whereas the ΔFaSdhC2 mutant exhibited resistance against most SDHIs. The transmembrane domains of FaSdhC1 are essential for regulating mycelial growth, virulence, and sensitivity to SDHIs. These findings provided valuable insights into how the two SdhC paralogues regulated the functional integrity of SDH, ROS homeostasis, and the sensitivity to SDHIs in phytopathogenic fungi.

The PbbHLH62/PbVHA-B1 module confers salt tolerance through modulating intracellular Na+/K+ homeostasis and reactive oxygen species removal in pear.

The vacuolar H+-ATPase (V-ATPase) is a multi-subunit membrane protein complex, which plays pivotal roles in building up an electrochemical H+-gradient across tonoplast, energizing Na+ sequestration into the central vacuole, and enhancing salt stress tolerance in plants. In this study, a B subunit of V-ATPase gene, PbVHA-B1 was discovered and isolated from stress-induced P. betulaefolia combining with RT-PCR method. The RT-qPCR analysis revealed that the expression level of PbVHA-B1 was upregulated by salt, drought, cold, and exogenous ABA treatment. Subcellular localization analyses showed that PbVHA-B1 was located in the cytoplasm and nucleus. Moreover, overexpression of PbVHA-B1 gene noticeably increased the ATPase activity and the tolerance to salt in transgenic Arabidopsis plants. In contrast, knockdown of PbVHA-B1 gene in P.betulaefolia by virus-induced gene silencing had reduced resistance to salt stress. In addition, using yeast one-hybride (Y1H) and yeast two-hybride (Y2H) screens, PbbHLH62, a bHLH transcription factor, was identified as a partner of the PbVHA-B1 promoter and protein. Then, we also found that PbbHLH62 positively regulate the expression of PbVHA-B1 and the ATPase activity after salt stress treatment. These findings provide evidence that PbbHLH62 played a critical role in the salt response. Collectively, our results demonstrate that a PbbHLH62/PbVHA-B1 module plays a positive role in salt tolerance by maintain intracellular ion and ROS homeostasis in pear.

PbARF19-mediated auxin signaling regulates lignification in pear fruit stone cells.

The stone cells in pear fruits cause rough flesh and low juice, seriously affecting the taste. Lignin has been demonstrated as the main component of stone cells. Auxin, one of the most important plant hormone, regulates most physiological processes in plants including lignification. However, the concentration effect and regulators of auxin on pear fruits stone cell formation remains unclear. Here, endogenous indole-3-acetic acid (IAA) and stone cells were found to be co-localized in lignified cells by immunofluorescence localization analysis. The exogenous treatment of different concentrations of IAA demonstrated that the application of 200 µM IAA significantly reduced stone cell content, while concentrations greater than 500 µM significantly increased stone cell content. Besides, 31 auxin response factors (ARFs) were identified in pear genome. Putative ARFs were predicted as critical regulators involved in the lignification of pear flesh cells by phylogenetic relationship and expression analysis. Furthermore, the negative regulation of PbARF19 on stone cell formation in pear fruit was demonstrated by overexpression in pear fruitlets and Arabidopsis. These results illustrated that the PbARF19-mediated auxin signal plays a critical role in the lignification of pear stone cell by regulating lignin biosynthetic genes. This study provides theoretical and practical guidance for improving fruit quality in pear production.

Filamentous morphology engineering of bacteria by iron metabolism modulation through MagR expression.

The morphology is the consequence of evolution and adaptation. Escherichia coli is rod-shaped bacillus with regular dimension of about 1.5 µm long and 0.5 µm wide. Many shape-related genes have been identified and used in morphology engineering of this bacteria. However, little is known about if specific metabolism and metal irons could modulate bacteria morphology. Here in this study, we discovered filamentous shape change of E. coli cells overexpressing pigeon MagR, a putative magnetoreceptor and extremely conserved iron-sulfur protein. Comparative transcriptomic analysis strongly suggested that the iron metabolism change and iron accumulation due to the overproduction of MagR was the key to the morphological change. This model was further validated, and filamentous morphological change was also achieved by supplement E. coli cells with iron in culture medium or by increase the iron uptake genes such as entB and fepA. Our study extended our understanding of morphology regulation of bacteria, and may also serves as a prototype of morphology engineering by modulating the iron metabolism.

M1 intestinal macrophages-derived exosomes promote colitis progression and mucosal barrier injury.

AIM: This work aimed to investigate the role of M1 intestinal macrophages-derived exosomes (M1-Exo) in colitis and its mechanism. METHODS: M1 polarization of intestinal macrophages was induced in vitro, and their exosomes were extracted and identified. Thereafter, the DSS-induced colitis mouse model was built. Each mouse was given intraperitoneal injection of exosomes, and then mouse weight and DAI were dynamically monitored. In addition, the levels of cytokines were detected by ELISA. After treatment with the TLR4 inhibitor Resatorvid, the effects of M1 macrophages-derived exosomes were observed. Besides, the mouse intestinal epithelial cells were cultured in vitro for observing function of M1-Exo. RESULTS: M1-exo aggravated the colitis and tissue inflammation in mice, activated the TLR4 signal, and destroyed the mucosal barrier. But M0 macrophages-derived exosomes (M0-Exo) did not have the above effects. Resatorvid treatment antagonized the roles of M1-exo. Moreover, as confirmed by cellular experiments in vitro, M1-exo destroyed mucosal barrier. CONCLUSION: M1-exo serve as the pro-inflammatory mediator, which can promote mouse colitis progression by activating TLR4 signal.

PuNDH9, a subunit of ETC Complex I regulates plant defense by interacting with PuPR1.

NAD+ and NADH play critical roles in energy metabolism, cell death, and gene expression. The NADH-ubiquinone oxidoreductase complex (Complex I) has been long known as a key enzyme in NAD+ and NADH metabolism. In the present study, we found and analyzed a new subunit of Complex I (NDH9), which was isolated from Pyrus ussuriensis combined with RT-PCR. Following infection with A. alternata, RT-qPCR analysis demonstrated an increase in the expression of PuNDH9. Genetic manipulation of PuNDH9 levels suggested that PuNDH9 plays key roles in NADH/NAD+ homeostasis, defense enzyme activities, ROS generation, cell death, gene expression, energy metabolism, and mitochondrial functions during the pear- A. alternata interaction. Furthermore, Y2H, GST-pull down, and a split-luciferase complementation imaging assays revealed that PuNDH9 interacts with PuPR1. We discover that PuNDH9 and PuPR1 synergistically activate defense enzyme activities, ROS accumulation, cell death, and plant defenses. Collectively, our findings reveal that PuNDH9 is likely important for plant defenses.

The natural polycyclic tetramate macrolactam HSAF inhibit Fusarium graminearum through altering cell membrane integrity by targeting FgORP1.

Fusarium graminearum is a dominant phytopathogenic fungus causing Fusarium head blight (FHB) in cereal crops. Heat-stable antifungal factor (HSAF) is a polycyclic tetramate macrolactam (PoTeM) isolated from Lysobacter enzymogenes that exhibits strong antifungal activity against F. graminearum. HSAF significantly reduces the DON production and virulence of F. graminearum. Importantly, HSAF exhibited no cross-resistance to carbendazim, phenamacril, tebuconazole and pydiflumetofen. However, the target protein of HSAF in F. graminearum is unclear. In this study, the oxysterol-binding protein FgORP1 was identified as the potential target of HSAF using surface plasmon resonance (SPR) combined with RNA-sequence (RNA-seq). The RNA-seq results showed cell membrane and ergosterol biosynthesis were significantly impacted by HSAF in F. graminearum. Molecular docking showed that HSAF binds with arginine 1205 and glutamic acid 1212, which are located in the oxysterol-binding domain of FgORP1. The two amino acids in FgORP1 are responsible for HSAF resistance in F. graminearum though site-directed mutagenesis. Furthermore, deletion of FgORP1 led to significantly decreased sensitivity to HSAF. Additionally, FgORP1 regulates the mycelial growth, conidiation, DON production, ergosterol biosynthesis and virulence in F. graminearum. Overall, our findings revealed the mode of action of HSAF against F. graminearum, indicating that HSAF is a promising fungicide for controlling FHB.

HIPK2 mediates M1 polarization of microglial cells via STAT3: A new mechanism of depression-related neuroinflammation.

This study aimed to investigate the role of protein kinase HIPK2 in depression and its associated mechanism. The chronic unpredictable mild stress (CUSM) model was constructed to simulate mice with depression to detect the mouse behaviors. Moreover, by using mouse microglial cells BV2 as the model. After conditional knockdown of HIPK2, the depressive behavior disorder of mice was improved, meanwhile, neuroinflammation was alleviated, and the M1 cell proportion was reduced. Similar results were obtained after applying the HIPK2 inhibitor tBID or ASO-HIPK2 treatment. HIPK2 was overexpressed in BV2 cells, which promoted M1 polarization of cells, while tBID suppressed the effect of HIPK2 and reduced the M1 polarized level in BV2 cells. Pull-down assay results indicated that HIPK2 bound to STAT3 and promoted STAT3 phosphorylation. We found that HIPK2 can bind to STAT3 to promote its phosphorylation, which accelerates M1 polarization of microglial cells, aggravates the depressive neuroinflammation, and leads to abnormal behaviors. HIPK2 is promising as the new therapeutic target of depression.

PbHsfC1a-coordinates ABA biosynthesis and H2O2 signalling pathways to improve drought tolerance in Pyrus betulaefolia.

Abiotic stresses have had a substantial impact on fruit crop output and quality. Plants have evolved an efficient immune system to combat abiotic stress, which employs reactive oxygen species (ROS) to activate the downstream defence response signals. Although an aquaporin protein encoded by PbPIP1;4 is identified from transcriptome analysis of Pyrus betulaefolia plants under drought treatments, little attention has been paid to the role of PIP and ROS in responding to abiotic stresses in pear plants. In this study, we discovered that overexpression of PbPIP1;4 in pear callus improved tolerance to oxidative and osmotic stresses by reconstructing redox homeostasis and ABA signal pathways. PbPIP1;4 overexpression enhanced the transport of H2O2 into pear and yeast cells. Overexpression of PbPIP1;4 in Arabidopsis plants mitigates the stress effects caused by adding ABA, including stomatal closure and reduction of seed germination and seedling growth. Overexpression of PbPIP1;4 in Arabidopsis plants decreases drought-induced leaf withering. The PbPIP1;4 promoter could be bound and activated by TF PbHsfC1a. Overexpression of PbHsfC1a in Arabidopsis plants rescued the leaf from wilting under drought stress. PbHsfC1a could bind to and activate AtNCED4 and PbNCED4 promoters, but the activation could be inhibited by adding ABA. Besides, PbNCED expression was up-regulated under H2O2 treatment but down-regulated under ABA treatment. In conclusion, this study revealed that PbHsfC1a is a positive regulator of abiotic stress, by targeting PbPIP1;4 and PbNCED4 promoters and activating their expression to mediate redox homeostasis and ABA biosynthesis. It provides valuable information for breeding drought-resistant pear cultivars through gene modification.

Corrigendum: Double-negative T cells regulate hepatic stellate cell activation to promote liver fibrosis progression via NLRP3.

[This corrects the article DOI: 10.3389/fimmu.2022.857116.].

Construction and Validation of a Prognostic Model Based on Pyroptosisrelated Genes in Bladder Cancer.

BACKGROUND: Bladder cancer (BCa) is a highly prevalent disease with a poor prognosis. There is no better forecasting method for it yet. Current studies demonstrate that pyroptosis is involved in the development and progression of various cancers. METHODS: This study employed bioinformatics techniques to analyze the data of BCa patients obtained from the TCGA and GEO databases in order to construct a prognostic risk model. The TCGA dataset was used for the training set, and the multiple external datasets (including GSE13507, GSE31684, GSE48075, IMvigor210, and GSE32894) were applied as the validation sets. Prognostic-associated pyroptosis genes screened by univariate Cox regression analysis were utilized to construct the lasso Cox regression model. GO and KEGG analysis results identified the selected genes that are primarily involved in the inflammation and cell death processes. The related patients were grouped into low- and high-risk groups. Kaplan-Meier survival analysis was performed to compare survival differences between the risk groups. The accuracy of this risk prediction model was assessed by ROC. We also applied the Human Protein Atlas (HPA) to detect the protein expression of these genes. Subsequently, qRT-PCR was performed to verify the expression of these model genes. RESULTS: There are 29 pyroptosis-related genes with significant expression differences between BCa and corresponding adjacent tissues, and 11 genes (SH2D2A, CHMP4C, MRFAP1L1, GBP2, EHBP1, RAD9A, ANXA1, TMEM109, HEYL, APOL2, ORMDL1) were picked by univariate and LASSO Cox regression analysis. Immunological cell infiltration and ssGSEA results further indicated that the low and high-risk groups were substantially correlated with the immune status of BCa patients. According to TCGA and multiple external datasets, Kaplan-Meier survival curves showed the overall survival rate of the high-risk group to be decreased. ROC curves showed the model established to be accurate and reliable. Moreover, the HPA database also demonstrated the verification of the modeled genes' expression in BCa and normal bladder tissue using the HPA database. qRT-PCR results also suggested the up-regulated EHBP1 and down-regulated RAD9A mRNA expression levels to be confirmed in 15 pairs of BCa and corresponding adjacent tissues. CONCLUSION: This study presents the development and validation of a novel gene signature associated with pyroptosis, which holds the potential for predicting patient outcomes in BCa and providing insights into the immune microenvironment of BCa.

Paeoniflorin suppresses neuronal ferroptosis to improve the cognitive behaviors in Alzheimer's disease mice.

Aim of this research was to examine the impact of paeoniflorin (Pae) in suppressing the occurrence of ferroptosis in individuals with Alzheimer's disease (AD). The study utilized APP/PS1 mice with AD as the experimental subjects. Following the administration of Pae, the cognitive behaviors of mice were evaluated and the key indexes of ferroptosis were measured, as well as levels of oxidative stress (OS). For in-vitro experiments, Erastin was adopted for inducing the ferroptosis of PC12 cells, and the level of cell ferroptosis was detected after Pae treatment. Pae improved the cognitive ability of AD mice, reduced the level of ferroptosis, decreased the iron ion and MAD levels in brain tissues, and increased SOD expression. In PC12 cells, Pae suppressed the Erastin-induced ferroptosis, mitigated oxidative damage, and reduced the level of ROS. Based on the findings from our research, it was observed that Pae exhibited a specific binding affinity to P53, leading to the suppression of ferroptosis. This mechanism ultimately resulted in the improvement of nerve injury in mice with AD.

Protection of a novel velvet antler polypeptide PNP1 against cerebral ischemia-reperfusion injury.

AIM: We isolated a novel polypeptide PNP1 from velvet antler and investigated the role of PNP1 in ischemia reperfusion and its associated mechanism. METHODS: We built the ischemia reperfusion mouse model by the middle cerebral artery occlusion (MCAO) approach. Thereafter, PNP-1 was injected via the tail vein, and neurological function was scored. Meanwhile, the tissue injury level was detected through hematoxylin & eosin (HE) and immunohistochemical (IHC) staining, inflammatory factor levels were determined with enzyme-linked immunosorbent assay (ELISA), while protein levels through Western blotting. In addition, vascular endothelial cells were used to construct the oxygen-glucose deprivation (OGD) injury model in vitro, so as to detect the intervention effect of PNP1 on endothelial injury. Additionally, microglial cells were utilized to construct the inflammatory injury model to examine the impact of PNP1 on the polarization of microglial cells. RESULTS: PNP1 suppressed hypoxic cerebral injury in MCAO mice, decreased the tissue inflammatory factors, promoted tissue angiogenesis, and reduced the ischemic penumbra area. Experimental results in vitro demonstrated that, PNP1 suppressed vascular endothelial cell injury, and inhibited microglial M1 polarization as well as inflammatory response. CONCLUSION: Velvet antler polypeptide PNP1 isolated in this study has the anti-ischemic cerebral injury effect, and its mechanism is associated with suppressing vascular endothelial cell injury and microglial cell inflammatory response.