Current status and breakthroughs in treating advanced non-small cell lung cancer with EGFR exon 20 insertion mutations.

Recently, targeted therapy and immunotherapy have emerged as effective treatment options for non-small cell lung cancer (NSCLC). This progress has been facilitated by the rapid development of diagnostic and therapeutic technologies and the continuous research and development of new drugs, leading to a new era in precision medicine for NSCLC. This is a breakthrough for patients with common mutations in the human epidermal growth factor receptor (EGFR) gene in NSCLC. Consequently, the use of targeted drugs has significantly improved survival. Nevertheless, certain rare genetic mutations are referred to as EGFR exon 20 insertion (ex20ins) mutations, which differ in structure from conventional EGFR gene mutations, namely, exon 19 deletion mutations (19-Del) and exon 21 point mutations. Owing to their distinct structural characteristics, patients harboring these EGFR ex20ins mutations are unresponsive to traditional tyrosine kinase inhibitor (TKI) therapy. This particular group of patients did not fall within the scope of their applicability. However, the activating A763_Y764insFQEA mutation elicits a more pronounced response than mutations in the near and far regions of the C-helix immediately following it and should, therefore, be treated differently. Currently, there is a lack of effective treatments for EGFR ex20ins mutations NSCLC. The efficacy of chemotherapy has been relatively favorable, whereas the effectiveness of immunotherapy remains ambiguous owing to inadequate clinical data. In addition, the efficacy of the first- and second-generation targeted drugs remains limited. However, third-generation and novel targeted drugs have proven to be effective. Although novel EGFR-TKIs are expected to treat EGFR ex20ins mutations in patients with NSCLC, they face many challenges. The main focus of this review is on emerging therapies that target NSCLC with EGFR ex20ins and highlight major ongoing clinical trials while also providing an overview of the associated challenges and research advancements in this area.

Correlation between serum inflammatory factors and cognitive function in patients with high-altitude polycythemia: A case-control study.

The purpose of this study is to investigate the serum inflammatory factors in patients with high-altitude polycythemia (HAPC) and their correlation with cognitive function. The subjects were recruited and placed into a HAPC group and control group. Serum samples were collected, and inflammatory factors (interleukin-1beta [IL-1ß], monocyte chemoattractant protein-1 [MCP-1], and tumor necrosis factor-alpha [TNF-α]) were measured using ELISA kits. The mini-mental State Examination (MMSE) was used to assess cognitive function. According to the MMSE scores, HAPC group was further divided into normal cognitive function group (HNCF) and cognitive dysfunction group (HCDF). In comparison with the control group, the MMSE scores in the HAPC group were significantly low (P < .05), whereas the serum levels of IL-1ß, MCP-1, and TNF-α were significantly high (P < .01). Among the HAPC group (n = 60), 21 belonged to the HCDF and 39 belonged to the HNCF. Compared with the HNCF, the IL-1ß, MCP-1, and TNF-α in the HCDF were significantly increased (P < .01). The Pearson correlation analysis showed that inflammatory factors were positively correlated with hemoglobin, and negatively correlated with MMSE. Serum inflammatory cytokines IL-1, MCP-1, and TNF-α were increased in HAPC, and HAPC exhibited cognitive dysfunction. Considering chronic hypoxia environment influences the change of the red blood cell metabolic and inflammatory factor, red blood cells and inflammatory factor in plateau is likely to be affected by patients with vascular lesions, increase cognitive impairment.

Discovery of cinnamylaldehyde-derived mono-carbonyl curcumin analogs as anti-gastric cancer agents via suppression of STAT3 and AKT pathway.

The structural modification of curcumin has always been a hotspot in drug development. In this paper, a class of cinnamylaldehyde-derived mono-carbonyl curcumin analogs (MCAs) with 7-carbon-links were designed and synthesized and their anticancer properties were evaluated. Through screening anti-gastric cancer activity of these compounds, H1 exhibited the strongest cytotoxic activity by inhibiting cell viability and colony formation, inducing cell cycle G2/M phase arrest in vitro (SGC-7901 and AGS gastric cancer cells). Moreover, the SGC-7901 subcutaneous tumor-bearing mice studies revealed that H1 significantly inhibited the tumor growth of gastric cancer. We explored the possible potential targets of H1 through network pharmacology. Mechanistically, our results demonstrated that H1 showed potential anti-gastric cancer activity through suppression of the STAT3 and AKT signaling pathway in vitro and in vivo, which was validated by molecular docking. Overall, our results indicate the potential of H1 as a potent chemotherapeutic drug against gastric cancer.

Withaferin A, a natural thioredoxin reductase 1 (TrxR1) inhibitor, synergistically enhances the antitumor efficacy of sorafenib through ROS-mediated ER stress and DNA damage in hepatocellular carcinoma cells.

BACKGROUND: Sorafenib (Sora), a multi-target tyrosine kinase inhibitor, is widely recognized as a standard chemotherapy treatment for advanced hepatocellular carcinoma (HCC). However, drug resistance mechanisms hinder its anticancer efficacy. Derived from Withania somnifera, Withaferin A (WA) exhibits remarkable anti-tumor properties as a natural bioactive compound. This study aimed to examine the mechanisms that underlie the impacts of Sora and WA co-treatment on HCC. METHODS: Cell proliferation was evaluated through colony formation and MTT assays. Flow cytometry was employed to determine cellular apoptosis and reactive oxygen species (ROS) levels. The evaluation of apoptosis-related protein levels, DNA damage, and endoplasmic reticulum stress was conducte utilizing IHC staining and western blotting. Moreover, the caspase inhibitor Z-VAD-FMK, ATF4 siRNA, ROS scavenger N-acetyl cysteine (NAC), and TrxR1 shRNA were used to elucidate the underlying signaling pathways. To validate the antitumor effects of Sora/WA co-treatment, in vivo experiments were ultimately executed using Huh7 xenografts. RESULTS: Sora/WA co-treatment demonstrated significant synergistic antitumor impacts both in vivo and in vitro. Mechanistically, the enhanced antitumor impact of Sora by WA was achieved through the inhibition of TrxR1 activity, resulting in ROS accumulation. Moreover, ROS generation induced the activation of DNA damage and endoplasmic reticulum (ER) stress pathways, eventually triggering cellular apoptosis. Pre-treatment with the antioxidant NAC significantly inhibited ROS generation, ER stress, DNA damage, and apoptosis induced by Sora/WA co-treatment. Additionally, the inhibition of ATF4 by small interfering RNA (siRNA) attenuated Sora/WA co-treatment-induced apoptosis. In vivo, Sora/WA co-treatment significantly suppressed tumor growth in HCC xenograft models and decreased TrxR1 activity in tumor tissues. CONCLUSION: Our study suggests that WA synergistically enhances the antitumor effect of Sora, offering promising implications for evolving treatment approaches for HCC.

Successful avatrombopag combined with cyclosporine treatment for carboplatin/pegylated liposomal doxorubicin/bevacizumab-induced acquired amegakaryocytic thrombocytopenia in a patient with recurrent ovarian cancer: case report.

Carboplatin/pegylated liposomal doxorubicin/bevacizumab is an accepted standard anti-cancer treatment option for recurrent ovarian cancer. However, the occurrence of adverse events associated with this therapeutic regimen limits its continued clinical use. Among these adverse events, acquired amegakaryocytic thrombocytopenia is a rare but often potentially life-threatening adverse effect, and is intolerant to multiple treatment approaches. We report, for the first time, the successful treatment using avatrombopag combined with cyclosporine in one case of carboplatin/pegylated liposomal doxorubicin/bevacizumab-induced acquired amegakaryocytic thrombocytopenia, which was refractory or intolerant to glucocorticoids, intravenous immunoglobulin, recombinant human thrombopoietin, androgen, and even thrombopoietin receptor receptor agonist eltrombopag and herombopag. To date, this case manifests as normal platelet counts that are independent of transfusion. Our findings suggest that this combination is a potential and valuable alternative in acquired amegakaryocytic thrombocytopenia.

Design, synthesis, and evaluation of cyclic C7-bridged monocarbonyl curcumin analogs containing an o-methoxy phenyl group as potential agents against gastric cancer.

The structure-activity relationship (SAR) between toxicity and the types of linking ketones of C7 bridged monocarbonyl curcumin analogs (MCAs) was not clear yet. In the pursuit of effective and less cytotoxic chemotherapeutics, we conducted a SAR analysis using various diketene skeletons of C7-bridged MCAs, synthesized cyclic C7-bridged MCAs containing the identified low-toxicity cyclopentanone scaffold and an o-methoxy phenyl group, and assessed their anti-gastric cancer activity and safety profile. Most compounds exhibited potent cytotoxic activities against gastric cancer cells. We developed a quantitative structure-activity relationship model (R2 > 0.82) by random Forest method, providing important information for optimizing structure. An optimized compound 2 exhibited in vitro and in vivo anti-gastric cancer activity partly through inhibiting the AKT and STAT3 pathways, and displayed a favorable in vivo safety profile. In summary, this paper provided a promising class of MCAs and a potential compound for the development of chemotherapeutic drugs.

Nimbolide Exhibits Potent Anticancer Activity Through ROS-Mediated ER Stress and DNA Damage in Human Non-small Cell Lung Cancer Cells.

The non-small cell lung cancer (NSCLC) accounts for about 85% of all lung cancers. It is usually diagnosed at an advanced stage with poor prognosis. Nimbolide (NB), a terpenoid limonoid isolated from the flowers and leaves of neem tree, possesses anticancer properties in various cancer cell lines. However, the underlying mechanism of its anticancer effect on human NSCLC cells remains unclear. In the present study, we investigated the effect of NB on A549 human NSCLC cells. We found that NB treatment inhibits A549 cells colony formation in a dose-dependent manner. Mechanistically, NB treatment increases cellular reactive oxygen species (ROS) level, leading to endoplasmic reticulum (ER) stress, DNA damage, and eventually induction of apoptosis in NSCLC cells. Furthermore, all these effects of NB were blocked by pretreatment with antioxidant glutathione (GSH), the specific ROS inhibitor. We further knockdown CHOP protein by siRNA markedly reduced NB-induced apoptosis in A549 cells. Taken together, our findings reveal that NB is an inducer of ER stress and ROS; these findings may contribute to increasing the therapeutic efficiency of NSCLC.

Structural Optimization Design of Dual Robot Gripper Unloading Device Based on Intelligent Optimization Algorithms and Generative Design.

The main aim of this paper is to explore new approaches to structural design and to solve the problem of lightweight design of structures involving multivariable and multi-objectives. An integrated optimization design methodology is proposed by combining intelligent optimization algorithms with generative design. Firstly, the meta-model is established to explore the relationship between design variables, quality, strain energy, and inherent energy. Then, employing the Non-dominated Sorting Genetic Algorithm III (NSGA-III), the optimal frameworks of the structure are sought within the entire design space. Immediately following, a structure is rebuilt based on the principle of cooperative equilibrium. Furthermore, the rebuilt structure is integrated into a generative design, enabling automatic iteration by controlling the initial parameter set. The quality and rigidity of the structure under different reconstructions are evaluated, resulting in solution generation for structural optimization. Finally, the optimal structure obtained is validated. Research outcomes indicate that the quality of structures generated through the comprehensive optimization method is reduced by 27%, and the inherent energy increases by 0.95 times. Moreover, the overall structural deformation is less than 0.003 mm, with a maximum stress of 3.2 MPa-significantly lower than the yield strength and meeting industrial usage standards. A qualitative study and analysis of the experimental results substantiate the superiority of the proposed methodology for optimized structural design.

Efficacy and Safety of Drug-Eluting Bead TACE in the Treatment of Primary or Secondary Liver Cancer.

Background: The drug-eluting beads transarterial chemoembolization (DEB-TACE) has already been used in hepatic malignancies. We aim to evaluate the efficacy and safety of DEB-TACE in treating primary or secondary liver cancer. Methods: We retrospectively evaluated 59 patients with hepatic malignancies, including 41 patients with primary liver cancer and 18 patients with secondary liver cancer, between September 2016 and February 2019. All patients were treated with DEB-TACE. Objective response rate (ORR) and disease control rate (DCR) were evaluated by mRECIST. The pain was assessed using a numerical rating scale (NRS) where 0 represented no pain, and a score of ten was unbearable. Adverse reactions were assessed according to Common Terminology Criteria for Adverse Events 4.0 (CTCAE4.0). Results: In the subgroup of primary liver cancer, 3 patients (7.32%) got complete response, 13 patients (31.71%) got partial response, 21 patients (51.22%) experienced stable disease, and 4 patients (9.76%) suffered progressive disease; ORR was 39.02% and DCR was 90.24%. In the subgroup of secondary liver cancer, 0 patients (0%) got complete response, 6 patients (33.33%) got partial response, 11 patients (61.11%) experienced stable disease, and 1 patient (5.56%) suffered progressive disease; ORR was 33.33% and DCR was 94.44%. We did not find any difference when comparing the efficacy between primary and secondary liver cancer (P=0.612). The one-year survival rate was 70.73% for primary liver cancer and 61.11% for secondary liver cancer. There was no significant difference between the two groups (P=0.52). For the patients with CR or PR, no factor could predict the efficacy of DEB-TACE. The most common treatment-related adverse reactions were short-term liver function disorders. The symptoms included fever (20.34%), abdomen pain (16.95%), and vomiting (5.08%), all patients with adverse reactions got remission after treatment. Conclusions: DEB-TACE has a promising effect in the treatment of primary or secondary liver cancer. The treatment-related adverse reactions are tolerable.

Curcumin protects against cerebral ischemia-reperfusion injury in rats by attenuating oxidative stress and inflammation: A meta-analysis and mechanism exploration.

Accumulating evidence has suggested that curcumin may protect against cerebral ischemia-reperfusion injury (CIRI). However, biological mechanisms vary across studies, limiting the clinical applicability of these findings. We performed a meta-analysis on publications evaluating curcumin administration in rat models of CIRI. Furthermore, we sought to test the hypothesis that curcumin alleviates CIRI through diminishing oxidation and inflammation. We searched PubMed, Embase, Web of Science, and Cochrane from the starting date of each database to May 2022 for experimental rat studies exploring the use of curcumin after ischemia reperfusion. Included articles were assessed for bias using SYRCLE's risk of bias tool. Data were aggregated by a random effects model. Curcumin administration significantly reduced neurological deficit score (20 studies; pooled mean difference [MD] = -1.57; 95% CI, -1.78 to -1.36, P < .00001), infarct volume (18 studies; pooled MD = -17.56%; 95% CI, -20.92% to -14.20%; P < 0.00001), and brain water content (8 studies, pooled MD = -11.29%, 95% CI: -16.48%, -6.11%, P < .00001). Compared with control, the levels of superoxide dismutase, glutathione, and glutathione peroxidase were significantly higher, whereas the levels of reactive oxygen species, malondialdehyde, interleukin-1ß, interleukin-6, interleukin-8, and nuclear factor kappa B were significantly lower (P < .05). Subgroup analysis raised the possibility that intervention affections differed by curcumin's dose. To our knowledge, this is the first meta-analysis of curcumin's neuroprotection and mechanisms in rat CIRI models. Our analysis suggests the neuroprotective potential of curcumin in CIRI via antioxidant activity and anti-inflammatory effect. More research is required to further confirm the effectiveness and safety of curcumin on ischemic stroke therapy.

Pyroptosis: A new insight of non-small-cell lung cancer treatment.

Non-small cell lung cancer (NSCLC) has become one of the most common malignant tumors. Emerging evidence has shown that tumor resistance to apoptosis by damaging or bypassing apoptotic cell death is a major contributor to poor responses to therapy in patients with NSCLC. Pyroptosis is a new type of cytolytic and inflammatory programmed death distinct from apoptosis. Currently, pyroptosis has been reported to cause a strong inflammatory response and significant tumor suppression. It is considered a promising therapeutic strategy and prognosis for NSCLC. In this review, we summarized the characteristics of pyroptosis from its underlying basis and role in NSCLC, thereby providing the potential of pyroptosis as a therapeutic strategy and highlighting the challenges of activating pyroptosis in NSCLC treatment.

MicroRNA-25-5p negatively regulates TXNIP expression and relieves inflammatory responses of brain induced by lipopolysaccharide.

Sepsis is one of the most common causes of death in patients suffering from severe infection or injury. Currently, a specific effective therapy remains to be established. In the present study, miR-25-5p, miR-105, miR-106b-5p, miR-154-3p, miR-20b-5p, miR-295-3p, miR-291-3p, miR-301b, miR-352, and miR-93-5p were predicted to target TXNIP mRNA from the databases of miRDB, Targetscan, and microT-CDS. The luciferase reporter assay confirmed that miR-25-5p negatively regulates TXNIP expression. The ELISA analyses and western blotting demonstrated that miR-25-5p downregulated the production of IL-1ß, IL-6, IL-8, and TNF-α in lipopolysaccharide (LPS)-stimulated cells or rats, as well as the protein levels of TXNIP, NLRP3, and cleaved caspase-1. In addition, miR-25-5p increased the cell viability and decreased the apoptosis in LPS-stimulated CTX TNA2 cells and reduced the abnormal morphology of the brain in LPS-stimulated rats. Besides, miR-25-5p decreased the relative mean fluorescence intensity of DCF in LPS-stimulated CTX TNA2 cell, apoptosis, and protein levels of MnSOD and catalase in LPS-stimulated brains. These findings indicate that miR-25-5p downregulated LPS-induced inflammatory responses, reactive oxygen species production, and brain damage, suggesting that miR-25-5p is a candidate treatment for septic encephalopathy.

Erratum: Author correction to 'Design, synthesis and biological evaluation of chalcone analogues with novel dual antioxidant mechanisms as potential anti-ischemic stroke agents' [Acta Pharmaceutica Sinica B 9 (2019) 335-350].

[This corrects the article DOI: 10.1016/j.apsb.2019.01.003.].

A rare case of recurrent primary dumbbell-shaped spinal hydatidosis.

Spinal hydatidosis, which affects the thoracic vertebrae, is not only an extremely rare occurrence, but is also characterized by a high recurrence rate. Here, we reported a case of 67-years-old man who presented with recurrent spinal hydatid disease. The condition was originally misdiagnosed as Schwannoma via medical imaging, but eventually confirmed by postoperative pathology. He was subjected to surgery, combined with adjuvant drug therapy. Unfortunately, he experienced recurrent spinal hydatid disease and had to undergo hydatid cyst excision in over 5 years.

Slice imputation: Multiple intermediate slices interpolation for anisotropic 3D medical image segmentation.

We introduce a novel frame-interpolation-based method for slice imputation to improve segmentation accuracy for anisotropic 3D medical images, in which the number of slices and their corresponding segmentation labels can be increased between two consecutive slices in anisotropic 3D medical volumes. Unlike previous inter-slice imputation methods, which only focus on the smoothness in the axial direction, this study aims to improve the smoothness of the interpolated 3D medical volumes in all three directions: axial, sagittal, and coronal. The proposed multitask inter-slice imputation method, in particular, incorporates a smoothness loss function to evaluate the smoothness of the interpolated 3D medical volumes in the through-plane direction (sagittal and coronal). It not only improves the resolution of the interpolated 3D medical volumes in the through-plane direction but also transforms them into isotropic representations, which leads to better segmentation performances. Experiments on whole tumor segmentation in the brain, liver tumor segmentation, and prostate segmentation indicate that our method outperforms the competing slice imputation methods on both computed tomography (1\% Dice improvement for CT liver tumor segmentation) and magnetic resonance images volumes (over 2\% Dice improvement for MRI prostate segmentation) in most cases.

Anti-Inflammatory Effect of Ginsenoside Rg1 on LPS-Induced Septic Encephalopathy and Associated Mechanism.

BACKGROUND: Sepsis frequently occurs in patients after infection and is highly associated with death. Septic encephalopathy is characterized by dysfunction of the central nervous system, of which the root cause is a systemic inflammatory response. Sepsis-associated encephalopathy is a severe disease that frequently occurs in children, resulting in high morbidity and mortality. OBJECTIVES: In the present study, we aimed to investigate the neuroprotective mechanism of ginsenoside Rg1 in response to septic encephalopathy. METHODS: Effects of ginsenoside Rg1 on septic encephalopathy were determined by cell viability, cytotoxicity, ROS responses, apoptosis assays, and histological examination of the brain. Inflammatory activities were evaluated by expression levels of IL-1ß, IL-6, IL-10, TNF-α, and MCP-1 using qPCR and ELISA. Activities of signaling pathways in inflammation were estimated by the production of p-Erk1/2/Erk1/2, p-JNK/JNK, p-p38/p38, p-p65/p65, and p-IkBα/IkBα using western blot. RESULTS: LPS simulation resulted in a significant increase in cytotoxicity, ROS responses, and apoptosis and a significant decrease in cell viability in CTX TNA2 cells, as well as brain damage in rats. Moreover, the production of IL-1ß, IL-6, IL-10, TNF-α, and MCP-1 was reported to be significantly stimulated in CTX TNA2 cells and the brain, confirming the establishment of in vitro and in vivo models of septic encephalopathy. The damage and inflammatory responses induced by LPS were significantly decreased by treatment with Rg1. Western blot analyses indicated that Rg1 significantly decreased the production of p-Erk1/2/Erk1/2, p-JNK/JNK, p-p38/p38, p-p65/p65, and p- IkBα/IkBα in LPS-induced CTX TNA2 cells and brain. CONCLUSION: These findings suggested that Rg1 inhibited the activation of NF-κB and MAPK signaling pathways, which activate the production of proinflammatory cytokines and chemokines. The findings of this study suggested that ginsenoside Rg1 is a candidate treatment for septic encephalopathy.

Molecular mechanisms and therapeutic relevance of gasdermin E in human diseases.

Gasdermin E (GSDME) is one of the main members of the GSDM family and is originally involved in hereditary hearing loss. Recent studies have reported that GSDME expression is epigenetically silenced by methylation in several common tumours, thereby enhancing tumour proliferation and metastasis. GSDME is also downregulated in cancer tissues compared with normal tissues, which suggests that GSDME can be considered a tumour suppressor. Furthermore, GSDME is the effector protein of caspase-3 and granzyme B in pyroptosis, and it plays a significant role in innate immunity, tissue damage, cancer, and hearing loss, thus revealing potential novel therapeutic avenues. A great deal of evidence reveals that GSDME can be implemented as a biomarker in cancer diagnosis and monitoring, chemotherapy, immunotherapy, and chemoresistance. Based on the current knowledge of GSDME, this review is focussed on its mechanism of action and the most recent advances in its role in cancer and normal physiology.

Optimization and feedback control system of dilution rate for 1,3-propanediol in two-stage fermentation: A theoretical study.

In utilizing glycerol to produce 1,3-propanediol by microbial fermentation, the problems of low utilization rate and poor production performance need to be addressed. Based on the analysis of a mathematical model for 1,3-propanediol production from glycerol by Klebsiella pneumoniae, this study theoretically investigated the effects of the dilution rate and the initial glycerol concentration in a two-stage fermentation process and the feasibility of applying the feedback control methods. First, the optimal operation conditions of initial glycerol concentration and dilution rate were obtained. Through the use of feedback control theory, a control strategy for dilution rate was designed and optimized to shorten the settling time (time required for fermentation to reach stability) from 60.92 to 36.68 h for the first reactor, and from 53.66 to 22.68 h for the second reactor. In addition, the yield of 1,3-propanediol in both two reactors reached up to 0.5 g·g-1 . The simulation results indicated that the feedback control strategy for dilution rate increased the product concentration, reduced the residual glycerol in the fermentation broth, and greatly improved the performance of the fermentation. A feeding strategy of automatic control for dilution rate has been established and will be applied as an effective guiding scheme in automatic continuous fermentations for production of 1,3-propanediol.

Targeting Reactive Oxygen Species Capacity of Tumor Cells with Repurposed Drug as an Anticancer Therapy.

Accumulating evidence shows that elevated levels of reactive oxygen species (ROS) are associated with cancer initiation, growth, and response to therapies. As concentrations increase, ROS influence cancer development in a paradoxical way, either triggering tumorigenesis and supporting the proliferation of cancer cells at moderate levels of ROS or causing cancer cell death at high levels of ROS. Thus, ROS can be considered an attractive target for therapy of cancer and two apparently contradictory but virtually complementary therapeutic strategies for the regulation of ROS to treat cancer. Despite tremendous resources being invested in prevention and treatment for cancer, cancer remains a leading cause of human deaths and brings a heavy burden to humans worldwide. Chemotherapy remains the key treatment for cancer therapy, but it produces harmful side effects. Meanwhile, the process of de novo development of new anticancer drugs generally needs increasing cost, long development cycle, and high risk of failure. The use of ROS-based repurposed drugs may be one of the promising ways to overcome current cancer treatment challenges. In this review, we briefly introduce the source and regulation of ROS and then focus on the status of repurposed drugs based on ROS regulation for cancer therapy and propose the challenges and direction of ROS-mediated cancer treatment.