**5. Conclusions**

We have shown increased levels of amino acid and protein hydroperoxides measured in the serum of asthmatic patients, using a recently developed technically undemanding real-time CBA assay. Furthermore, estimated oxidative stress was related to BAL and blood inflammatory biomarkers, spirometry values, and CT airway geometry measures, but not asthma severity or histological indices of airway remodeling. More prospective and experimental studies are needed to verify the biological role of increased circulating oxidative stress in the clinical course of asthma and extrapulmonary complications.

**Supplementary Materials:** The following supporting information can be downloaded at https: //www.mdpi.com/article/10.3390/biomedicines10071499/s1. Table S1: Clinical characteristics of asthmatic patients divided into mild, moderate, and severe disease stages, including airway imaging and histo(cyto)logy.

**Author Contributions:** Conceptualization, S.B.-S. and K.W.; funding acquisition, S.B.-S.; patient recruitment, S.B.-S., M.C.-L. and K.W.; CBA assay, J.P., M.O., T.S. and K.W.; bronchoscopy, J.S.; lung CT, J.Z.; histopathological examination, K.O.; validation, S.B.-S., M.S., J.G.B. and B.J.; CBA assay parameters analysis, L.Z. and J.G.B.; statistical Analysis, L.Z.; writing—original draft preparation, S.B.-S., B.J., M.C.-L. and D.P.P.; writing—review, critical revision, and final approval, all authors. All authors have read and agreed to the published version of the manuscript.

**Funding:** This project was funded by the National Science Centre, Poland, based on decision No. DEC-2013/09/B/NZ5/00758 (to S.B.-S.).

**Institutional Review Board Statement:** The study was conducted in accordance with the Declaration of Helsinki, and the protocol was approved by the Ethics Committee of the Jagiellonian University approved the study (approval number: KBET/151/B/2013).

**Informed Consent Statement:** Informed consent was obtained from all subjects involved in the study.

**Data Availability Statement:** The data presented in this study are available upon request from the corresponding author. The data are not publicly available due to patients' origin.

**Conflicts of Interest:** The authors declare no conflict of interest.


*2.4. Auto-Segmentation Models*

We compared the 3D, 2.5D, and 2D approaches (Figure 1) across three segmentation models: capsule networks (CapsNets) [23], UNets [24], and nnUNets [22]. These models are considered the highest-performing auto-segmentation models in the biomedical domain [9,22,23,25–29]. The 3D models process a 3D patch of the image as input, all feature maps and parameter tensors in all layers are 3D, and the model output is the segmented 3D patch of the image. Conversely, 2D models process a 2D slice of the image as input, all feature maps and parameter tensors in all layers are 2D, and the model output is the segmented 2D slice of the image. The 2.5D models process five consecutive slices of the image as input channels. The remaining parts of the 2.5D model, including the feature maps and parameter tensors, are 2D, and the model output is the segmented 2D middle slice among the five slices. We did not develop 2.5D nnUNets, because the self-configuring paradigm of nnUNets was developed for 3D and 2D inputs but not for 2.5D inputs. Notably, the aim of training and testing nnUNets (in addition to UNets) was to ensure that our choices of hyperparameters did not cause one approach (such as 3D) to perform better than other approaches. The nnUNet can self-configure the best hyperparameters for the 3D and 2D approaches but not for the 2.5D approach. As a result, we did not train or test 2.5D nnUNets. The model architectures are described in Supplementary Material S4.

#### *2.5. Training*

We trained the CapsNet and UNet models for 50 epochs using Dice loss and the Adam optimizer [30]. Initial learning rate was set at 0.002. We used dynamic paradigms for learning rate scheduling, with a minimal learning rate of 0.0001. The hyperparameters for our CapsNet and UNet models were chosen based on the model with the lowest Dice loss over the validation set. The hyperparameters for the nnUNet model were self-configured by the model [22]. Supplementary Material S5 describes the training hyperparameters for CapsNet and UNet.

#### *2.6. Performance Metrics*

For each model (CapsNet, UNet, and nnUNet), we compared the performance of 3D, 2.5D, and 2D approaches using the following metrics: (1) Segmentation accuracy: we used the Dice score to quantify the segmentation accuracy of the fully trained models over the test set.31 We compared Dice scores between the three approaches for three representative anatomic structures of the brain: the third ventricle, thalamus, and hippocampus. The mean Dice scores for the auto-segmentation of these brain structures are reported together with their 95% confidence interval. To compute the 95% confidence interval for each Dice score, we used bootstrapping to sample the 114 Dice scores over the test set, with replacement, 1000 times. We then calculated the mean Dice score for each of the 1000 samples, giving us 1000 mean Dice scores. We then sorted these mean Dice scores and found the range that covered 95% of them, which is equivalent to the 95% confidence interval for each Dice score. (2) Performance when training data is limited: we trained the models using the complete training set and random subsets of the training set with 600, 240, 120, and 60 MR images. The models trained on these subsets were then evaluated over the test

set. (3) Computational speed during training: we compared the time needed to train the 3D, 2.5D, and 2D models per training example per epoch until the model converged. (4) Computational speed for segmenting an MR image: we compared how quickly each of the 3D, 2.5D, and 2D models segment one brain MRI volume. (5) Computational memory: we compared how much GPU memory is required, in units of megabytes, to train and deploy each of the 3D, 2.5D, and 2D models.

**Figure 1.** We compared three segmentation approaches: 3D, 2.5D, and 2D. The 2D approach analyzes and segments one slice of the image, the 2.5D approach analyzes five consecutive slices of the image to segment the middle slice, and the 3D approach analyzes and segments a 3D volume of the image.