Mast Cells Initiate Type 2 Inflammation through Tryptase Released by MRGPRX2/MRGPRB2 Activation in Atopic Dermatitis.

Atopic dermatitis (AD) is a common chronic inflammatory skin disease characterized by T helper 2 inflammation as the core pathogenic mechanism. MRGPRX2 plays a key role in nonhistamine allergies and neuroimmune mechanisms in chronic inflammatory dermatitis. However, the role of MRGPRX2 in AD and the development of type 2 inflammation is not yet clear. This study aimed to define the role of MRGPRX2 in type 2 inflammation development and cytokine release in AD by determining its levels in patients with AD and healthy controls. Furthermore, MrgprB2-conditional knockout (MrgprB2-/-) and wild-type mice were used to construct an MC903-induced AD mouse model to observe skin inflammation and cytokine release. Tryptase and its antagonist were applied separately to MrgprB2-/- mice with AD and wild-type mice with AD to confirm the role of the MRGPRB2-tryptase axis in the development of type 2 inflammation in AD. We found that AD severity and type 2 cytokine levels were not associated with IgE levels but were associated with MRGPRX2/MRGPRB2 expression. MrgprB2-/- mice with AD showed milder phenotypes and inflammatory infiltration in the skin than wild-type mice with AD. Tryptase released by MRGPRX2/MRGPRB2 activation is involved in the release of type 2 cytokines, which contributes to inflammatory development in AD.

Robot versus video-assisted thoracoscopic thymectomy for large thymic epithelial tumors: a propensity-matched analysis.

BACKGROUND: Both video-assisted thoracoscopic surgery (VATS) thymectomy and robot-assisted thoracoscopic surgery (RATS) thymectomy have been suggested as technically sound approaches for early-stage thymic epithelial tumors. However, the choice of VATS or RATS thymectomy for large and advanced thymic epithelial tumors remains controversial. In this study, the perioperative outcomes of VATS and RATS thymectomy were compared in patients with large thymic epithelial tumors (size ≥5.0 cm). METHODS: A total of 113 patients with large thymic epithelial tumors who underwent minimally invasive surgery were included. Sixty-three patients underwent RATS, and 50 patients underwent VATS. Patient characteristics and perioperative variables were compared. RESULTS: Compared with the VATS group, the RATS group experienced a shorter operation time (median: 110 min vs.130 min; P < 0.001) and less blood loss (30.00 ml vs. 100.00 ml, P < 0.001). No patients in the RATS group needed conversion to open surgery, but in the VATS series, five patients required conversion to open procedures (0% vs. 14.29%, P = 0.054). The rate of concomitant resection in the RATS group was similar to that in the VATS group (11.43% vs. 5.71%; P = 0.673). There was no significant difference between the two groups in the duration of chest tube (P = 0.587), postoperative complications (P = 1.000), and the duration of postoperative hospital stay (P = 0.141). CONCLUSION: For large thymic epithelial tumors, RATS thymectomy can be performed safely and effectively in a radical fashion. Due to the advanced optics and precise instrument control, concomitant resections can be easily achieved in larger thymic epithelial tumors using the robotic approach.

Viaminate Inhibits Propionibacterium Acnes-induced Abnormal Proliferation and Keratinization of HaCat Cells by Regulating the S100A8/S100A9- MAPK Cascade.

BACKGROUND: Viaminate, a vitamin A acid drug developed in China, has been clinically used in acne treatment to regulate epithelial cell differentiation and proliferation, inhibit keratinization, reduce sebum secretion, and control immunological and anti-inflammatory actions; however, the exact method by which it works is unknown. METHODS: In the present study, acne was induced in the ears of rats using Propionibacterium acnes combined with sebum application. RESULTS: After 30 days of treatment with viaminate, the symptoms of epidermal thickening and keratin overproduction in the ears of rats were significantly improved. Transcriptomic analysis of rat skin tissues suggested that viaminate significantly regulated the biological pathways of cellular keratinization. Gene differential analysis revealed that the S100A8 and S100A9 genes were significantly downregulated after viaminate treatment. The results of qPCR and Western blotting confirmed that viaminate inhibited the expression of S100A8 and S100A9 genes and proteins in rat and HaCat cell acne models, while its downstream pathway MAPK (MAPK p38/JNK/ERK1/2) protein expression levels were suppressed. Additional administration of the S100A8 and S100A9 complex protein significantly reversed the inhibitory effect of viaminate on abnormal proliferation and keratinization levels in acne cell models. CONCLUSION: In summary, viaminate can improve acne by modulating S100A8 and S100A9 to inhibit MAPK pathway activation and inhibit keratinocyte proliferation and keratinization levels.

Successful secukinumab therapy in plaque psoriasis is associated with altered gut microbiota and related functional changes.

Introduction: The role of gut microbiome dysbiosis in the pathogenesis of psoriasis has gained increasing attention in recent years. Secukinumab, targeting interleukin (IL)-17, has a promising efficacy in psoriasis treatment. However, it remains unclear the gut microbiota alteration and related functional changes caused by successful secukinumab therapy in psoriatic patients. Methods: In our study, we compared the fecal microbiome profile between psoriatic patients after secukinumab successful treatment (AT) and the other two groups, psoriatic patients without therapy (BT) and healthy people (H), respectively, by using next-generation sequencing targeting 16S ribosomal RNA. Then, shotgun metagenomic sequencing was first used to characterize bacterial gut microbial communities and related functional changes in the AT group. Results: We found that the diversity and structure of the microbial community in the AT group were significantly changed compared to those in the BT group and the H group. The AT group showed a microbiota profile characterized by increased proportions of the phylum Firmicute, families Ruminococcaceae, and a reduction in the phylum Bacteroidota (elevated F/B ratio). To detect functional alteration, we discovered that secukinumab treatment may construct a more stable homeostasis of the gut microbiome with functional alteration. There were different KEGG pathways, such as the downregulated cardiovascular diseases pathway and the upregulated infectious diseases in the AT group. By metagenomic analysis, the metabolic functional pathway was changed after secukinumab therapy. Discussion: It seems that gut microbiota investigation during biologic drug treatment is useful for predicting the efficacy and risks of drug treatment in disease.

Effect of initiative pulmonary bullectomy on the risk of post-operative pneumothorax in patients with esophageal carcinoma: a propensity score-matched analysis.

Background: Postoperative pneumothorax can lead to additional invasive intervention and extended hospitalization. The effect of initiative pulmonary bullectomy (IPB) during the esophagectomy on preventing postoperative pneumothorax remains controversial. This study evaluated the efficacy and safety of IPB in patients who underwent minimally invasive esophagectomy (MIE) for esophageal carcinoma complicated by ipsilateral pulmonary bullae. Methods: Data from 654 consecutive patients with esophageal carcinoma who underwent MIE from January 2013 to May 2020 were retrospectively collected. A total of 109 patients who had a definite diagnosis of ipsilateral pulmonary bullae were recruited and classified into two groups: the IPB group and the control group (CG). Propensity score matching (PSM, match ratio =1:1), incorporating preoperative clinical features, was used to compare the perioperative complications and analyze efficacy and safety between IPB and control group. Results: The incidences of postoperative pneumothorax in the IPB and control groups was 3.13% and 40.63% respectively, with a significant difference (P<0.001). Logistic analyses indicated that removing ipsilateral bullae was associated with a lower risk (OR 0.030; 95% CI: 0.003-0.338; P=0.005) of incident postoperative pneumothorax. No significant difference was found between the two groups in terms of the incidence of anastomotic leakage (6.25% vs. 3.13%, P=1.000), arrhythmia (3.13% vs. 3.13%, P=1.000), chylothorax (0% vs. 3.13%, P=1.000) and other common complications. Conclusions: In esophageal cancer patients with ipsilateral pulmonary bullae, IPB performed in the same anesthesia process is an effective and safe method for the prevention of postoperative pneumothorax, allowing for a shorter postoperative rehabilitation time, and it does not exert unfavorable effects on complications.

Viaminate ameliorates Propionibacterium acnes-induced acne via inhibition of the TLR2/NF-κB and MAPK pathways in rats.

Viaminate, a retinoic acid derivative developed in China, has been clinically used for acne treatment to regulate and control keratinocyte cell differentiation and proliferation, inhibit keratinization, reduce sebum secretion, and regulate immune and anti-inflammatory functions; however, its potential molecular mechanism has not yet been elucidated. Therefore, we induced ear acne in rats using Propionibacterium acnes and sebum application. Symptoms of ear redness, epidermal thickening, inflammatory reaction, keratin overproduction, subcutaneous oil, and triglyceride (TG) accumulation improved significantly in acne model rats treated with viaminate for 30 days. Transcriptome analysis of rat skin tissues suggested that viaminate had significant regulatory effects on fatty acid metabolism and cellular keratinization pathways. Molecular target prediction suggested that toll-like receptor 2 (TLR2) may be a key target of viaminate's therapeutic mechanism. Western blotting results confirmed that viaminate inhibited the TLR2 and its downstream pathways, nuclear factor-kappa B (NF-κB) [NF-κB inhibitor alpha (IκBα)/NF-κB-p65] and mitogen-activated protein kinases (MAPKs) [MAPK p38/c-Jun N-terminal kinase (JNK)/extracellular regulated kinase 1/2 (ERK1/2)] in acne vulgaris rats. In vitro studies revealed that viaminate treatment attenuated P. acnes proliferation and P. acnes-induced inflammatory response in human keratinocytes and has an inhibitory effect on the activation of NF-κB and MAPKs, while overexpression of TLR2 attenuated these effects. In conclusion, viaminate ameliorates P. acnes-induced acne by inhibiting the proliferation and inflammatory response of keratinocytes, ascribed to the deactivation of the TLR2-mediated NF-κB and MAPK pathways.

Robot-assisted thymectomy in large anterior mediastinal tumors: A comparative study with video-assisted thymectomy and open surgery.

BACKGROUND: The aim of this study was to evaluate the safety and effectiveness of robot-assisted thymectomy (RAT) in large anterior mediastinal tumors (AMTs) (size ≥6 cm) compared with video-assisted thymectomy (VAT) and open surgery. METHODS: A total of 132 patients with large AMTs who underwent surgical resection from January 2016 to June 2022 were included in this study. A total of 61 patients underwent RAT, 36 patients underwent VAT and 35 patients underwent open surgery. Perioperative outcomes were compared. RESULTS: There were no significant differences in tumor size (p = 0.141), or pathological types (p = 0.903). Compared with the open group, the RAT and VAT groups were associated with a shorter operation time (115.00 vs. 160.00, p = 0.012; 122.50 vs. 160.00, p = 0.071), and less blood loss (50.00 vs. 200.00, p < 0.001; 50.00 vs. 200.00, p < 0.001), respectively. The rate of conversion in the RAT group was similar to that in the VAT group (6.56% vs. 13.89%, p = 0.229). Concomitant resection was less frequently performed in the VAT group than in the RAT and open groups (5.56% vs. 31.15%, p = 0.040; 5.56% vs. 31.43%, p = 0.006). VAT patients had a lower drainage volume (365.00 vs. 700.00 and 910.00 mL, p < 0.001), shorter duration of chest tube (2.00 vs. 3.00 and 4.00, p < 0.001), and shorter hospital stay (5.00 vs. 6.00 and 7.00, p < 0.001) than the RAT and open groups. There was no 30-day mortality in any group. No difference was seen in R0 resection rates (p = 0.846). The postoperative complication rates were similar among the three groups (p = 0.309). Total in-hospital costs (66493.90 vs. 33581.05 and 42876.40, p < 0.001) were significantly higher in the RAT group. CONCLUSIONS: RAT is safe and effective for the resection of large AMTs compared to VAT and open surgery. Vascular resection in RAT is technically feasible. A long-term follow-up is required.

Manipulating Metabolic Alterations and their Consequences to Unleash the Potential of Antitumor Immunotherapy.

Cellular metabolic reprogramming driven by oncogenic mutations is considered as a hallmark in the development of malignant cells, and has been a focus over the past decade. A common theme emerging from these metabolic alterations is that tumor cells can acquire necessary nutrients from a nutrient-limited microenvironment and utilize them to sustain growth and unrestrained cellular division. However, this significant metabolic flexibility and the hostile microenvironment caused by the insufficient vascular exchange, depletion of nutrients, hypoxia, and accumulation of waste products, can inhibit the metabolism and immune activity of tumor-infiltrating lymphocytes and impose barriers to effective antitumor immunotherapies. In this perspective, we review the classical alterations in tumorigenesis- associated metabolic reprogramming and examine the functional contribution of these aberrant metabolisms to the establishment and maintenance of an immunosuppressive microenvironment. Furthermore, we explore the possible approaches to targeting on these metabolic pathways to achieve antitumor immunotherapy, as well as some hypothetical or ongoing combination therapeutic strategies that could, to a certain extent, biologically rationalize and broaden the utility of immune checkpoint inhibitors. Ultimately, we elucidate some dietary modifications that can limit tumor-specific nutritional requirements and maximize the cytotoxicity of other antineoplastic drugs.

Efficient Generation of CRISPR/Cas9-Based Mutants Supported by Fluorescent Seed Selection in Different Arabidopsis Accessions.

Investigating the process of gamete formation in plants often requires the use of mutants of selected genes in various genetic backgrounds. For example, analysis of meiotic recombination based on sequencing or genotyping requires the generation of hybrids between two lines. Although T-DNA mutant collections of Arabidopsis thaliana are vast and easily accessible, they are largely confined to Col-0 background. This chapter describes how to efficiently generate knock-out mutants in different Arabidopsis accessions using CRISPR/Cas9 technology. The presented system is based on designing two single-guide RNAs (sgRNAs), which direct the Cas9 endonuclease to generate double-strand breaks at two sites, leading to genomic deletion in targeted gene. The presence of seed-expressed dsRed fluorescence cassette in the CRISPR construct facilitates preselection of genome-edited and transgene-free plants by monitoring the seed fluorescence under the epifluorescent microscope. The protocol provides the detailed information about all steps required to perform genome editing and to obtain loss-of-function mutants in different Arabidopsis accessions within merely two generations.

Effects of Glucose Metabolism, Lipid Metabolism, and Glutamine Metabolism on Tumor Microenvironment and Clinical Implications.

In recent years, an increasingly more in depth understanding of tumor metabolism in tumorigenesis, tumor growth, metastasis, and prognosis has been achieved. The broad heterogeneity in tumor tissue is the critical factor affecting the outcome of tumor treatment. Metabolic heterogeneity is not only found in tumor cells but also in their surrounding immune and stromal cells; for example, many suppressor cells, such as tumor-associated macrophages (TAMs), myeloid-derived suppressor cells (MDSCs), and tumor-associated T-lymphocytes. Abnormalities in metabolism often lead to short survival or resistance to antitumor therapy, e.g., chemotherapy, radiotherapy, targeted therapy, and immunotherapy. Using the metabolic characteristics of the tumor microenvironment to identify and treat cancer has become a great research hotspot. This review systematically addresses the impacts of metabolism on tumor cells and effector cells and represents recent research advances of metabolic effects on other cells in the tumor microenvironment. Finally, we introduce some applications of metabolic features in clinical oncology.

Effectiveness and safety of Adalimumab in psoriasis and its influence on gut microbiome.

Psoriasis is an inflammatory immune-mediated skin disease that significantly impacts physical and psychological well-being. Adalimumab (ADA), a tumor necrosis factor (TNF)-α antagonist, is used to treat psoriasis. This study was performed to assess the efficacy and safety of ADA, identify the fecal microbial composition of psoriasis patients, and explore the effect of ADA on the gut bacteria in psoriasis. Clinical characteristics of the 13 psoriasis patients before (BT) and after ADA treatment (AT) were collected. And total 39 fecal samples from 13 psoriasis patients (BT and AT) and 13 healthy controls were sequenced by 16S rRNA and analyzed by informatics methods. After three months' ADA treatment, physician global assessment (PGA), psoriasis area and severity index (PASI), dermatology life quality index (DLQI), state-trait anxiety inventory (STAI), and itch numeric rating scale (NRS) scores all decreased, and there were no severe adverse effects. Besides, the microbiota of the psoriasis group differed from that of the healthy group, but no microbial diversity and composition alteration were observed between psoriasis patients BT and AT. We suggested that the gut microbiome may change more slowly than skin lesions. Long-term follow-up of patients treated with ADA and further study of psoriasis based on microbiota may provide more evidence for the treatment of psoriasis.

Natural variation identifies SNI1, the SMC5/6 component, as a modifier of meiotic crossover in Arabidopsis.

The frequency and distribution of meiotic crossovers are tightly controlled; however, variation in this process can be observed both within and between species. Using crosses of two natural Arabidopsis thaliana accessions, Col and Ler, we mapped a crossover modifier locus to semidominant polymorphisms in SUPPRESSOR OF NPR1-1 INDUCIBLE 1 (SNI1), which encodes a component of the SMC5/6 complex. The sni1 mutant exhibits a modified pattern of recombination across the genome with crossovers elevated in chromosome distal regions but reduced in pericentromeres. Mutations in SNI1 result in reduced crossover interference and can partially restore the fertility of a Class I crossover pathway mutant, which suggests that the protein affects noninterfering crossover repair. Therefore, we tested genetic interactions between SNI1 and both RECQ4 and FANCM DNA helicases, which showed that additional Class II crossovers observed in the sni1 mutant are FANCM independent. Furthermore, genetic analysis of other SMC5/6 mutants confirms the observations of crossover redistribution made for SNI1 The study reveals the importance of the SMC5/6 complex in ensuring the proper progress of meiotic recombination in plants.

Oscillatory self-organization dynamics between soliton molecules induced by gain fluctuation.

In passively mode-locked fiber lasers (PMLFLs), the dissipative solitons (DSs) can self-organize to form complex structures through delicate interactions. However, it is still elusive to control these soliton structures by external influences. We here find that at a certain critical power, the location between two soliton molecules can be controlled by a slow modulated pump power. After applying the pump power with periodic fluctuation, two soliton molecules oscillate from the state of soliton molecular complex to stable distribution with maximum inter-molecular separation. During this process, the internal structure of each soliton molecule keeps steady. The slow gain depletion and recovery mechanism which plays a dominant role affects the motion of soliton molecules. These results could further expand the molecular analogy of spectroscopy and stimulate the development of optical information storage and processing.

The complete chloroplast genome of Viburnum dilatatum (Adoxaceae).

Viburnum dilatatum Thunb. is a large deciduous tree of Adoxaceae. In this study, the chloroplast genome sequence of V. dilatatum is 158,392 bp, consisting of a large single-copy (LSC) region with 87,070 bp, a small single-copy (SSC) region with 18,242 bp , and two inverted repeat (IR) regions with 26,540 bp. The GC content in the chloroplast genome of C. julianae is 38.1%. The chloroplast genome of V. dilatatum contains 126 genes, including 83 protein-coding genes, 39 tRNA genes, and 4 rRNA genes. Phylogenetic tree showed that V. dilatatum was clustered with V. utile.

MPMR: Multi-Scale Feature and Probability Map for Melanoma Recognition.

Malignant melanoma (MM) recognition in whole-slide images (WSIs) is challenging due to the huge image size of billions of pixels and complex visual characteristics. We propose a novel automatic melanoma recognition method based on the multi-scale features and probability map, named MPMR. First, we introduce the idea of breaking up the WSI into patches to overcome the difficult-to-calculate problem of WSIs with huge sizes. Second, to obtain and visualize the recognition result of MM tissues in WSIs, a probability mapping method is proposed to generate the mask based on predicted categories, confidence probabilities, and location information of patches. Third, considering that the pathological features related to melanoma are at different scales, such as tissue, cell, and nucleus, and to enhance the representation of multi-scale features is important for melanoma recognition, we construct a multi-scale feature fusion architecture by additional branch paths and shortcut connections, which extracts the enriched lesion features from low-level features containing more detail information and high-level features containing more semantic information. Fourth, to improve the extraction feature of the irregular-shaped lesion and focus on essential features, we reconstructed the residual blocks by a deformable convolution and channel attention mechanism, which further reduces information redundancy and noisy features. The experimental results demonstrate that the proposed method outperforms the compared algorithms, and it has a potential for practical applications in clinical diagnosis.

Comparative transcriptomic analysis of the flower induction and development of the Lei bamboo (Phyllostachys violascens).

BACKGROUND: Bamboo is a very important forest resource. However, the prolonged vegetative stages and uncertainty of flowering brings difficulties in bamboo flowers sampling. Until now, the flowering mechanism of bamboo is still unclear. RESULTS: In this study, three successive stages of flowering buds and the corresponding vegetative buds (non-flowering stage) from Lei bamboo (Phyllostachys violascens) were collected for transcriptome analysis using Illumina RNA-Seq method. We generated about 442 million clean reads from the above samples, and 132,678 unigenes were acquired with N50 of 1080 bp. A total of 7266 differentially expressed genes (DEGs) were determined. According to expression profile and gene function analysis, some environmental stress responsive and plant hormone-related DEGs were highly expressed in the inflorescence meristem formation stage (TF_1) while some floral organ development related genes were up-regulated significantly in floral organs determination stage (TF_2) and floral organs maturation (TF_3) stage, implying the essential roles of these DEGs in flower induction and maturation of Lei bamboo. Additionally, a total of 25 MADS-box unigenes were identified. Based on the expression profile, B, C/D and E clade genes were more related to floral organs development compared with A clade genes in Lei bamboo. CONCLUSIONS: This transcriptome data presents fundamental information about the genes and pathways involved in flower induction and development of Lei bamboo. Moreover, a critical sampling method is provided which could be benefit for bamboo flowering mechanism study.

Development and Characterization of EST-SSR Markers From RNA-Seq Data in Phyllostachys violascens.

Bamboo are woody grass species containing important economic and ecological values. Lei bamboo (Phyllostachys violascens) is a kind of shoot-producing bamboo species with the highest economic yield per unit area. However, identifying different varieties of Lei bamboo based on morphological characteristics is difficult. Microsatellites play an important role in plant identification and genetic diversity analysis and are superior to other molecular markers. In this study, we identified 18,356 expressed sequence tag-simple sequence repeat (EST-SSR) loci in Lei bamboo transcriptome data. A total of 11,264 primer pairs were successfully designed from unigenes of all EST-SSR loci, and 96 primer pairs were randomly selected and synthesized. A total of 54 primer pairs were used for classifying 16 Lei bamboo varieties and 10 different Phyllostachys species. The number of polymorphism alleles among the 54 primer pairs ranged from 3 to 12 for P. violascens varieties and 3 to 20 for Phyllostachys. The phylogenetic tree based on polymorphism alleles successfully distinguished 16 P. violascens varieties and 10 Phyllostachys species. Our study provides abundant EST-SSR resources that are useful for genetic diversity analysis and molecular verification of bamboo and suggests that SSR markers developed from Lei bamboo are more efficient and reliable than ISSR, SRAP or AFLP markers.

Baicalein protects human vitiligo melanocytes from oxidative stress through activation of NF-E2-related factor2 (Nrf2) signaling pathway.

Vitiligo is a complex disorder characterized by patchy loss of skin pigmentation due to abnormal melanocyte function. Overwhelming evidences have suggested that oxidative stress plays a major role in the loss of melanocytes thereby mediating the onset and progression of vitiligo. The nuclear factor erythroid 2-like factor 2 (Nrf2) is a master regulator of cellular redox homeostasis and the activation of Nrf2 signaling pathway is impaired in the vitiligo melanocytes. Baicalein, as flavonoid extracted from the Scutellaria baicalensis, has been proved to possess the ability to activate Nrf2 signaling pathway in other cell types and mouse model. Our previous data found that baicalein exerts a cytoprotective role in H2O2-induced apoptosis in human melanocytes cell line (PIG1). Based on these founding, we hypothesized that baicalein activates Nrf2 signaling pathway, alleviates H2O2-induced mitochondrial dysfunction and cellular damage, thereby protecting human vitiligo melanocytes from oxidative stress. In the present study, we found that baicalein effectively inhibited H2O2-induced cytotoxicity and apoptosis in human vitiligo melanocytes (PIG3V). Further results demonstrated that baicalein promoted Nrf2 nucleus translocation as well as up-regulated the expression of Nrf2 and its target gene, heme oxygenase-1 (HO-1). Moreover, the protective effects of baicalein against H2O2-induced cellular damage and apoptosis as well as mitochondrial dysfunction were abolished by Nrf2 knockdown. Additionally, we observed that Nrf2 knockdown suppressed proliferation and increased the sensitivity of PIG3V cells to H2O2 treatment. Finally, we explored the mechanism of baicalein associated with Nrf2 activation and found that the phosphorylation of Nrf2 as well as ERK1/2and PI3K/AKT signaling were not involved in the baicalein-induced activation of Nrf2. Taken together, these data clearly suggest that baicalein enhances cellular antioxidant defense capacity of human vitiligo melanocytes through the activation of the Nrf2 signaling pathway, providing beneficial evidence for the application of baicalein in the vitiligo treatment.