Tumor involvement of the trigone and urethra at the time of robot-assisted radical cystectomy is associated with adverse oncological outcomes.

INTRODUCTION: The trigone/urethra (T/U) has a distinct embryologic origin and a different histologic morphology compared to the rest of the urinary bladder. We sought to determine the association between tumors involved in the T/U and the presence of variant histology, pathologic, and oncologic outcomes in patients who underwent robot-assisted radical cystectomy (RARC). METHODS: Tumor location was classified into 2 groups: tumors in the bladder walls only, or tumors in the T/U area, with or without involvement of other bladder walls. Univariable and multivariable Cox regression models were used to determine the association between T/U with recurrence-specific (RSS), cancer-specific (CSS), and overall survival (OS). RESULTS: 608 patients who underwent RARC were identified, T/U involvement occurred in 191 (31%). Patients in the T/U group were more likely to have pT3/pT4 (57% vs. 42%, P < 0.01), positive surgical margins (21% vs. 9%, P < 0.01), and received salvage chemotherapy more frequently (16% vs. 8%, P < 0.01). Squamous variant histology was more frequent in the T/U group (25% vs. 17%, P = 0.02). On multivariable analysis, T/U location was independently associated with RSS (HR1.63, 95% CI 1.23-2.16, P < 0.01) and CSS (HR1.50, 95% CI 1.04-2.16, P = 0.02) but not OS. CONCLUSION: Residual T/U tumor involvement was associated with a higher risk of an advanced tumor stage, positive margin, cancer recurrence, and cancer-specific death.

Cluster formation tracking of networked perturbed robotic systems via hierarchical fixed-time neural adaptive approach.

This paper investigates the fixed-time cluster formation tracking (CFT) problem for networked perturbed robotic systems (NPRSs) under directed graph information interaction, considering parametric uncertainties, external perturbations, and actuator input deadzone. To address this complex problem, a novel hierarchical fixed-time neural adaptive control algorithm is proposed based on a hierarchical fixed-time framework and a neural adaptive control strategy. The objective of this study is to achieve accurate CFT of NPRSs within a fixed time. Specifically, we design a distributed observer algorithm to estimate the states of the virtual leader within a fixed time accurately. By using these observers, a neural adaptive fixed-time controller is developed in the local control layer to ensure rapid and reliable system performance. Through the use of the Lyapunov argument, we derive sufficient conditions on the control parameters to guarantee the fixed-time stability of NPRSs. The theoretical results are eventually validated through numerical simulations, demonstrating the effectiveness and robustness of the proposed approach.

Pharmacology and therapeutic potential of agarwood and agarwood tree leaves in periodontitis.

The main bioactive components of agarwood, derived from Aquilaria sinensis, include sesquiterpenes, 2-(2-phenethyl) chromone derivatives, aromatic compounds, and fatty acids, which typically exert anti-inflammatory, antioxidant, immune-modulating, hypoglycemic, and antitumor pharmacological effects in the form of essential oils. Agarwood tree leaves, rich in flavonoids, 2-(2-phenethyl) chromone compounds, and flavonoid compounds, also exhibit significant anti-inflammatory, antioxidant, and immune-modulating effects. These properties are particularly relevant to the treatment of periodontitis, given that inflammatory responses, oxidative stress, and immune dysregulation are key pathological mechanisms of the disease, highlighting the substantial potential of agarwood and agarwood tree leaves in this therapeutic area. However, the low solubility and poor bioavailability of essential oils present challenges that necessitate the development of improved active formulations. In this review, we will introduce the bioactive components, extraction methods, pharmacological actions, and clinical applications of agarwood and agarwood tree leaves, analyzing its prospects for the treatment of periodontitis.

Fungal Coculture of Herpotrichia sp. and Trametes versicolor Induces Production of Diverse Metabolites with Anti-Parkinson's Neuroprotective Activity.

Co-cultivation of isopod-associated fungi Herpotrichia sp. SF09 and Trametes versicolor SF09A led to the reciprocal induction of thirteen new compounds (1-7 and 9-13) with diverse architectures. Importantly, compounds 1 and 2 are rare fungal sesquiterpene-saccharide hybrids incorporating a xylopyranose moiety, compound (±)-3 represents the first example of a natural linear sesquiterpene racemate, and compound 7 is a rare α-pyrone derivative with a xylopyranose motif. Their structures were elucidated by analysis of NMR and mass spectrometry data, and their absolute configurations were determined by Mosher's method, microscale derivatization, and single-crystal X-ray diffraction, as well as ECD calculations. All the isolated compounds ameliorated MPP+-induced oxidative damage in PC12 cells in a dose-dependent fashion. Among them, compounds 5 and 15 showed significant protective action against neuronal injury by MPP+ at 5 µM. Meanwhile, transcriptome sequencing was performed to evaluate the molecular mechanism of the neuroprotective activity for compound 5. Results indicated that compound 5 might mitigate MPP+-induced neuronal injury through the regulation of multiple signaling pathways, including the PI3K-Akt and MAPK pathways. Our findings suggested that compound 5 could be a promising neuroprotective agent for the treatment of Parkinson's disease.

Robot-assisted uretero-enteric reimplantation for uretero-enteric anastomotic strictures following robot-assisted radical cystectomy: Surgical approach and outcomes over two decades.

Objective: We described the technique and outcomes of robot-assisted repair of uretero-enteric strictures (UES) following robot-assisted radical cystectomy (RARC) and urinary diversion. Methods: Retrospective review of our RARC database from November 2005 to August 2023 at Roswell Park Comprehensive Cancer center was performed. Patients who developed UES and ultimately underwent robot-assisted uretero-enteric reimplantation (RUER) were identified. Kaplan-Meier method was used to compute the cumulative incidence recurrence rate of UES after RUER. A multivariable regression model was used to identify variables associated with UES recurrence. Results: A total of 123 (15%) out of 808 RARC patients developed UES, of whom 52 underwent reimplantation (45 patients underwent RUER [n=55 cases] and seven patients underwent open uretero-enteric reimplantation). The median time from RARC to UES was 4.4 (interquartile range 3.0-7.0) months, and the median time between UES and RUER was 5.2 (interquartile range 3.2-8.9) months. The 3-year recurrence rate after RUER is about 29%. On multivariable analysis, longer hospital stay (hazard ratio 1.37, 95% confidence interval 1.16-1.61, p<0.01) was associated with recurrent UES after RUER. Conclusion: RUER for UES after RARC is feasible with durable outcomes although a notable subset of patients experienced postoperative complications and UES recurrence.

Isolation and biological activity of six new polyketide and terpenoid derivatives from Neopestalotiopsis Clavispora AL01.

A fungus strain, Neopestalotiopsis clavispora AL01, was isolated from the leaf spot of the plant Phoenix dactylifera. Further chemical investigation of the fermentation extract of this strain afforded six new secondary metabolites (1-6), along with 11 known compounds (7-17) which included a new natural compound (7). Their structures were determined by extensive spectroscopic analysis including one-and two-dimensional (1D and 2D) NMR spectroscopy, high-resolution electrospray ionization mass spectrometry (HRESIMS), and ECD and NMR calculations. All compounds were evaluated for their phytotoxic activities. Among them, compounds 10, 12 and 13 exhibited phytotoxic activities against Nicotiana tabacum. Compound 3 exhibited weak antibacterial activity against methicillin-resistant Staphylococcus aureus, Micrococcus luteus and Vibrio harveyi. Taken collectively, these findings establish a solid research foundation for future investigations on bioactive natural products derived from phytopathogenic fungi.

Does Ta Low-grade Urothelial Carcinoma of the Bladder With Focal High-grade Features Carry Worse Prognosis? The Roswell Park Comprehensive Cancer Center Experience.

OBJECTIVE: To describe the management and outcomes of patients with Ta predominantly low-grade urothelial carcinoma with focal high-grade features (FHG) (<5%), compared to those with Ta low grade (LG) and Ta high grade (HG). METHODS: Retrospective review of all patients who underwent transurethral resection of bladder tumor between 2005 and 2023. Patients with Ta disease were identified and categorized into LG, FHG, and HG. Kaplan Meier method was used to depict high-grade recurrence, T-stage progression, and radical cystectomy-free survival. RESULTS: Four hundred forty-nine patients with Ta disease were identified (LG 48%, FHG 12%, and HG 40%). Patients with FHG (32%) had a second-look transurethral resection of bladder tumor more frequently compared to LG (7%) and HG (29%) (P <.01). They received intravesical therapy more frequently compared to LG (36% vs 20%) but lower than HG (55%) (P <.01). They received radical cystectomy less frequently (7% compared to 20% for HG and 11% for LG, P = .01). HG recurrence-free survival at 1, 3, and 5years was HG (68%, 52%, and 43%), FHG (74%, 53%, and 49%), and LG (87%, 79%, and 73%) (log-rank P <.01). T progression-free survival at 1, 3, and 5years was HG (84%, 77%, and 70%), FHG (92%, 82%, and 82%), and LG (94%, 89%, and 85%) (log-rank P = .02). Cystectomy-free survival at 1, 3, and 5years was HG (92%, 84%, and 80%), FHG (96%, 94%, and 94%), and LG (99%, 95%, and 92%) (log-rank P <.01). CONCLUSION: Patients with Ta FHG seem to behave more like Ta HG disease in terms of high-grade recurrences, but they are less likely to experience T-stage progression and convert to cystectomy.

Is Stent-Free Ureteroenteric Anastomosis Associated With Lower Ureteroenteric Strictures After Robot-Assisted Radical Cystectomy and Ileal Conduit?

INTRODUCTION: We aimed to investigate the differences in perioperative outcomes, especially ureteroenteric strictures, between patients who underwent a stented ureteroenteric anastomosis at the time of robot-assisted radical cystectomy (RARC) and ileal conduit vs those who did not. METHODS: A retrospective review of our RARC database was performed (2009-2023). Patients were divided into those who received stented ureteroenteric anastomosis vs those who did not. Propensity score matching was performed in the ratio of 3 (stented ureteroenteric anastomosis) to 1 (stent-free) in terms of age, gender, BMI, race, American Society of Anesthesiologists score, neoadjuvant chemotherapy, Charlson Comorbidity Index, prior radiation therapy, previous abdominal surgery history, clinical T3/clinical T4 stage, preoperative metastasis, and preoperative hydronephrosis. A cumulative incidence curve was used to depict ureteroenteric strictures and a Cox regression model was used to identify variables associated with ureteroenteric strictures. RESULTS: Four hundred eighty-eight patients underwent RARC, 366 individuals underwent a stented ureteroenteric anastomosis, and 122 patients underwent a stent-free approach. There was no significant difference in 90-day overall complications, high-grade complications, readmissions, UTIs, leakage, and ileus (P > .05). Ureteroenteric strictures occurred at a rate of 13% and 18% at 1 and 2 years, respectively in the stented group, vs 7% and 10% in the stent-free group (P = .05). Stent placement was significantly associated with ureteroenteric strictures. CONCLUSIONS: Stent-free ureteroenteric anastomosis was associated with fewer strictures following RARC and ileal conduit.

Bi-2-(2-phenethyl)chromone derivatives from agarwood of Aquilaria filaria in the Philippines.

Twelve undescribed 2-(2-phenethyl)chromone dimers (1-12) were isolated from EtOAc extract of agarwood originating from Aquilaria filaria in the Philippines, guided by a UHPLC-MS analysis. Their structures were elucidated by 1D NMR, 2D NMR, and HR-ESI-MS spectra. The absolute configuration of 2-(2-phenylethyl)chromone dimers was determined by single-crystal X-ray diffraction analysis and comparison of the experimental and calculated ECD spectra. Compounds 1, 2, 5 and 9-12 exhibited potent to moderate anti-inflammatory activity with IC50 values in the range of 22.43 ± 0.86 to 53.88 ± 4.06 µM.

Estimation of the Band Gap of Carbon Nanotube Bundles.

The electronic structure of carbon nanotube bundles (CNTBs) can be a tough task for the routine first-principle calculation. The difficulty comes from several issues including the atomic structure, the boundary condition, and above all the very large number of atoms that makes the calculation quite cumbersome. In this work, we estimated the band gap of the CNTBs based on the results from single-walled carbon nanotubes (SWCNTs) under different deformations. The effects of squeezing, stretching, and torsion on the bands of SWCNTs were investigated through first-principle calculations, from which the band gaps of bundles were analyzed because the effects of these deformations were qualitatively independent when the distortions were small. Specifically, the gaps of (4,4) and (8,0) CNTBs under a reasonable torsional strength were predicted, wherein we were able to see metal-semiconductor and semiconductor-metal transitions, respectively. Such reversible mechanical modification of the conductivity may be helpful to the future band-gap engineering in nanoscale circuits.

The role of neoadjuvant chemotherapy for patients with variant histology muscle invasive bladder cancer undergoing robotic cystectomy: Data from the International Robotic Cystectomy Consortium.

OBJECTIVE: To assess the role of neoadjuvant chemotherapy (NAC) before robot-assisted radical cystectomy (RARC) for patients with variant histology (VH) muscle-invasive bladder cancer (MIBC). METHODS: Retrospective review of 988 patients who underwent RARC (2004-2023) for MIBC. Primary outcomes included the utilization of NAC among this cohort of patients, frequency of downstaging, and discordance between preoperative and final pathology in terms of the presence of VH. Secondary outcomes included disease-specific (DSS), recurrence-free (RFS), and overall survival (OS). RESULTS: A total of 349 (35%) had VH on transurethral resection or at RARC. The 4 most common VH subgroups were squamous (n = 94), adenocarcinoma (n = 64), micropapillary (n = 34), and sarcomatoid (n = 21). There was no difference in OS (log-rank: P = 0.43 for adenocarcinoma, P = 0.12 for micropapillary, P = 0.55 for sarcomatoid, P = 0.29 for squamous), RFS (log-rank: P = 0.25 for adenocarcinoma, P = 0.35 for micropapillary, P = 0.83 for sarcomatoid, P = 0.79 for squamous), or DSS (log-rank P = 0.91 for adenocarcinoma, P = 0.15 for micropapillary, 0.28 for sarcomatoid, P = 0.92 for squamous) among any of the VH based on receipt of NAC. Patients with squamous histology who received NAC were more likely to be downstaged on final pathology compared to those who did not (P < 0.01). CONCLUSION: Our data showed no significant difference in OS, RFS, or DSS for patients with VH MIBC cancer who received NAC before RARC. Patients with the squamous variant who received NAC had more pathologic downstaging compared to those who did not. The role of NAC among patients with VH is yet to be defined. Results were limited by small number in each individual group and lack of exact proportion of VH.

Unveiling CRESS DNA Virus Diversity in Oysters by Virome.

Oysters that filter feed can accumulate numerous pathogens, including viruses, which can serve as a valuable viral repository. As oyster farming becomes more prevalent, concerns are mounting about diseases that can harm both cultivated and wild oysters. Unfortunately, there is a lack of research on the viruses and other factors that can cause illness in shellfish. This means that it is harder to find ways to prevent these diseases and protect the oysters. This is part of a previously started project, the Dataset of Oyster Virome, in which we further study 30 almost complete genomes of oyster-associated CRESS DNA viruses. The replication-associated proteins and capsid proteins found in CRESS DNA viruses display varying evolutionary rates and frequently undergo recombination. Additionally, some CRESS DNA viruses have the capability for cross-species transmission. A plethora of unclassified CRESS DNA viruses are detectable in transcriptome libraries, exhibiting higher levels of transcriptional activity than those found in metagenome libraries. The study significantly enhances our understanding of the diversity of oyster-associated CRESS DNA viruses, emphasizing the widespread presence of CRESS DNA viruses in the natural environment and the substantial portion of CRESS DNA viruses that remain unidentified. This study's findings provide a basis for further research on the biological and ecological roles of viruses in oysters and their environment.

Development of performance and learning rate evaluation models in robot-assisted surgery using electroencephalography and eye-tracking.

The existing performance evaluation methods in robot-assisted surgery (RAS) are mainly subjective, costly, and affected by shortcomings such as the inconsistency of results and dependency on the raters' opinions. The aim of this study was to develop models for an objective evaluation of performance and rate of learning RAS skills while practicing surgical simulator tasks. The electroencephalogram (EEG) and eye-tracking data were recorded from 26 subjects while performing Tubes, Suture Sponge, and Dots and Needles tasks. Performance scores were generated by the simulator program. The functional brain networks were extracted using EEG data and coherence analysis. Then these networks, along with community detection analysis, facilitated the extraction of average search information and average temporal flexibility features at 21 Brodmann areas (BA) and four band frequencies. Twelve eye-tracking features were extracted and used to develop linear random intercept models for performance evaluation and multivariate linear regression models for the evaluation of the learning rate. Results showed that subject-wise standardization of features improved the R2 of the models. Average pupil diameter and rate of saccade were associated with performance in the Tubes task (multivariate analysis; p-value = 0.01 and p-value = 0.04, respectively). Entropy of pupil diameter was associated with performance in Dots and Needles task (multivariate analysis; p-value = 0.01). Average temporal flexibility and search information in several BAs and band frequencies were associated with performance and rate of learning. The models may be used to objectify performance and learning rate evaluation in RAS once validated with a broader sample size and tasks.

Chemical constituents of the marine-derived fungus Acremonium sp. AN-13.

Two new compounds named 3(S)-hydroxy-1-(2,4,5-trihydroxy-3,6- dimethylphenyl)-hex-4E-en-1-one (1) and acremonilactone (2), together with nine known compounds (3-11), were isolated from the fermentation broth of Acremonium sp. associated with marine sediments collected from South China Sea. NMR and HRESIMS spectroscopic analysis elucidated the structure of two new compounds. Compound 2 had characteristic rotary gate shape skeleton with a six-membered lactone. Compounds 1 and 9 showed DPPH radical scavenging activity with inhibition rates of 96.50 and 85.95% at the concentration of 0.5 mg/ml, respectively. Moreover, compounds 4, 6 and 11 showed definite antibacterial activity against Staphylococcus aureus ATCC 6538.

Sesquiterpenoids and bibenzyl derivative from Dendrobium hercoglossum.

Three new sesquiterpenoids, dendrohercoglin A - C (1-3), and one new bibenzyl derivative, dendronbiline D (4), together with nine known sesquiterpenoids (5-13) were isolated from Dendrobium hercoglossum. The structures of the new compounds were elucidated by extensive spectroscopic analysis as well as NMR and ECD calculations. All the compounds were evaluated for their neuroprotective and anti-inflammatory activities. Compounds 2 and 3 increased the H2O2-damaged SH-SY5Y cell viabilities from 43.3% to 58.6% and 68.4%, respectively. Compound 4 exhibited pronounced anti-inflammatory activity with IC50 value of 9.5 ± 0.45 µM which was superior to the reference compound quercetin (IC50: 15.7 ± 0.89 µM).

The impact of NCCN-compliant multidisciplinary conference on the uptake of active surveillance among eligible patients with localized prostate cancer.

INTRODUCTION: We sought to investigate the impact of National Comprehensive Cancer Network (NCCN)-compliant multidisciplinary conference on the uptake of active surveillance (AS) among eligible patients with prostate cancer. METHODS: Retrospective review of our AS database was performed. Patients who are eligible for AS who sought a second opinion at a comprehensive cancer center (2010-2021) were presented to the multidisciplinary Localized Prostate Cancer Conference (LPCC) that includes urologists, radiation oncologists, pathologists, and patient advocates. Cochrane Armitage test was used to examine trends over time. Multivariable regression models were fit to evaluate variables associated with the receipt of AS. RESULTS: Seven hundred twelve patients were identified (19% NCCN very low risk, 32% low risk, and 49% intermediate favorable risk). 43% were recommended AS as the preferred option by the community compared to 68% by LPCC, and 65% elected AS. Recommending AS significantly increased between 2010 and 2021 by the community (from 26% to 57%) and by LPCC (from 52% to 82%), while the proportion of men who received AS increased from 47% to 80% during the same period (P < 0.0001 for all). More recent LPCC era 2017 to 2021 (OR 12.31, 95% CI, 5.60-27.03, P < 0.0001), African American race (OR 0.42, 95% CI, 0.18-0.96, P = 0.04), positive cores at biopsy (OR 0.96, 95% CI, 0.94-0.97, P < 0.0001), age (OR 1.14, 95% CI, 1.10-1.18, P < 0.0001), NCCN low risk (OR 0.25, 95% CI, 0.08-0.81, P = 0.02) and NCCN intermediate favorable risk (OR 0.03, 95% CI, 0.01-0.09, P < 0.0001) were associated with receipt of AS. CONCLUSION: AS recommendation increased significantly over time by community urologists and to a higher extent by NCCN-compliant multidisciplinary conference. The Uptake of AS significantly increased within the same period. More recent LPCC era 2017 to 2021, African American race, the proportion of positive cores at biopsy, age, and NCCN risk were the main determinants of receipt of AS.

Does the urinary microbiome profile change after treatment of bladder cancer?

INTRODUCTION: We sought to investigate the change in the urinary microbiome profile after transurethral resection of bladder tumor (TURBT). METHODS: Urine specimens were collected from consecutive patients with bladder cancer. Patients were divided into those with bladder tumors ("Tumor group": de novo tumors or recurrent/progressed after TURBT ± intravesical therapy) versus those without evidence of recurrence after treatment "No Recurrent Tumor group". Samples were analyzed using 16S rRNA sequencing. Alteration in the urinary microbiome was described in terms of alpha (diversity within a sample measured by Observed, Chao, Shannon, and Simpson indices), beta diversities (diversity among different samples measured by Brady Curtis Diversity index), and differential abundance of bacteria at the genus level. Analyses were adjusted for gender, method of preservation (frozen vs preservative), and method of collection (mid-stream vs. catheter). RESULTS: Sixty-eight samples were analyzed (42 in "Tumor" vs 26 in "No Recurrent Tumor" groups). The median age was 70 years (IQR 64-74) and 85% were males. All patients in the "No Recurrent Tumor" group had non-muscle invasive bladder cancer and 85% received BCG compared to 69% and 43% for the "Tumor" group, respectively. There was no significant difference in alpha diversity (p > 0.05). Beta diversity was significantly different (p = 0.04). Veillonella and Bifidobacterium were more abundant in the "Tumor" group (> 2FC, p = 0.0002), while Escherichia-Shigella (> 2FC, p = 0.0002) and Helococcus (> 2FC, p = 0.0008) were more abundant in the "No Recurrent Tumor" group. CONCLUSION: Bladder cancer patients with no recurrence and/or progression exhibited a different urinary microbiome profile compared to those with tumors.

Developing cognitive workload and performance evaluation models using functional brain network analysis.

Cognition, defined as the ability to learn, remember, sustain attention, make decisions, and solve problems, is essential in daily activities and in learning new skills. The purpose of this study was to develop cognitive workload and performance evaluation models using features that were extracted from Electroencephalogram (EEG) data through functional brain network and spectral analyses. The EEG data were recorded from 124 brain areas of 26 healthy participants conducting two cognitive tasks on a robot simulator. The functional brain network and Power Spectral Density features were extracted from EEG data using coherence and spectral analyses, respectively. Participants reported their perceived cognitive workload using the SURG-TLX questionnaire after each exercise, and the simulator generated actual performance scores. The extracted features, actual performance scores, and subjectively assessed cognitive workload values were used to develop linear models for evaluating performance and cognitive workload. Furthermore, the Pearson correlation was used to find the correlation between participants' age, performance, and cognitive workload. The findings demonstrated that combined EEG features retrieved from spectral analysis and functional brain networks can be used to evaluate cognitive workload and performance. The cognitive workload in conducting only Matchboard level 3, which is more challenging than Matchboard level 2, was correlated with age (0.54, p-value = 0.01). This finding may suggest playing more challenging computer games are more helpful in identifying changes in cognitive workload caused by aging. The findings could open the door for a new era of objective evaluation and monitoring of cognitive workload and performance.

Classification of Color Fundus Photographs Using Fusion Extracted Features and Customized CNN Models.

This study focuses on overcoming challenges in classifying eye diseases using color fundus photographs by leveraging deep learning techniques, aiming to enhance early detection and diagnosis accuracy. We utilized a dataset of 6392 color fundus photographs across eight disease categories, which was later augmented to 17,766 images. Five well-known convolutional neural networks (CNNs)-efficientnetb0, mobilenetv2, shufflenet, resnet50, and resnet101-and a custom-built CNN were integrated and trained on this dataset. Image sizes were standardized, and model performance was evaluated via accuracy, Kappa coefficient, and precision metrics. Shufflenet and efficientnetb0demonstrated strong performances, while our custom 17-layer CNN outperformed all with an accuracy of 0.930 and a Kappa coefficient of 0.920. Furthermore, we found that the fusion of image features with classical machine learning classifiers increased the performance, with Logistic Regression showcasing the best results. Our study highlights the potential of AI and deep learning models in accurately classifying eye diseases and demonstrates the efficacy of custom-built models and the fusion of deep learning and classical methods. Future work should focus on validating these methods across larger datasets and assessing their real-world applicability.