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Automated qEEG Case Study Generation with Retrieval-Augmented AI and Clinical Data Integration

3 Replies

DOI: 10.31038/JCRM.2025831

Abstract

Quantitative electroencephalography (qEEG) offers objective biomarkers of brain function across neuropsychiatric conditions, but clinical EEG case reports are traditionally labor-intensive to produce. We describe a reproducible Python-based pipeline that automatically processes raw BrainVision EEG data, extracts spectral qEEG features, integrates patient clinical scores (e.g. Brief Psychiatric Rating Scale, BPRS), retrieves relevant literature via Europe PMC, and uses a retrieval augmented large language model (RAG-LLM) to generate structured narrative case reports. EEG preprocessing (filtering, artifact removal, referencing) and feature computation (power in delta, theta, alpha, beta bands, etc.) are implemented using open-source MNE-Python tools in a BIDS-compliant [1,2] framework. Patient metadata such as age, diagnosis, and BPRS severity provide clinical context alongside EEG features (e.g. the known increase in theta power and theta/beta ratio in schizophrenia) [3]. Key EEG findings are combined with dynamically retrieved evidence from Europe PMC – an open access repository of ~36 million biomedical abstracts and 5 million full-text articles – to ground the report in up-to-date knowledge [4]. Using a RAG-LLM approach, the system formulates context-aware prompts that guide the model to cite recent studies and summarize findings. For example, prior work has shown retrieval-augmented LLMs significantly improve accuracy in clinical question answering [5] compared to base models, and in dedicated frameworks (e.g. EEG-MedRAG) unify EEG domain [6] knowledge and patient data for diagnostic guidance. Our pipeline yields a draft case report that mimics the structure of a clinician’s report: background, methods, results (EEG summary and clinical scores), and an evidence-supported discussion.

Keywords

qEEG, RAG, Clinical Data, EEG Reports, AI Neuropsychiatry, EEG Preprocessing, Spectral Features, BPRS, Case Automation, Neuroinformatics

Introduction

EEG remains an indispensable tool in neuroscience and psychiatry, providing noninvasive recordings of brain activity. Quantitative EEG (qEEG) – the analysis of EEG power spectrum bands – has been studied [3] as a putative biomarker in disorders such as schizophrenia, ADHD, depression and bipolar disorder [7]. For example, schizophrenia is often associated with increased delta/theta and reduced alpha [3] power. Clinical context is captured by symptom scales like the BPRS, which quantify severity of [3] psychiatric symptoms and are routinely collected in research studies. Integrating EEG features with clinical metrics can enhance interpretation (e.g. correlating theta increase with BPRS depression subscore). However, manually generating a cohesive case report that synthesizes EEG analyses with relevant literature is time-consuming and subjective.

Recent advances in large language models (LLMs) and retrieval-augmented generation (RAG) offer a new paradigm: an AI-driven pipeline can automatically assemble multimodal data and external knowledge into an explanatory narrative. LLMs have demonstrated capability in medical domains, but [5,8] they are prone to hallucination unless grounded by real data. RAG addresses this by coupling an LLM with a knowledge base: the model retrieves pertinent documents (here from Europe PMC) and [9] conditions its output on this evidence. Studies in healthcare have shown that RAG-augmented systems yield more accurate, up-to-date answers than base LLMs alone. For instance [5,8] Masanneck et al. tested multiple LLMs on neurology guidelines and found that a fixed-document RAG setup markedly improved accuracy over unfettered models, though caution remains for hallucinations and case-based scenarios. Similarly, Kuo et al. describe a hierarchical RAG pipeline that retrieves [5] heterogeneous clinical trial data and generates reports with higher factual consistency and greatly [8,10] reduced authoring time compared to manual methods. Inspired by such successes, we propose applying RAG to qEEG.

EEG Data Preprocessing and Spectral Feature Extraction

Raw EEG data (e.g. BrainVision.eeg/.vhdr files) are ingested and organized according to the Brain Imaging Data Structure (BIDS) for neurophysiology. We use MNE-Python to apply a standard [1] preprocessing chain: band-pass filtering (e.g. 1–50 Hz), removal of line noise, and artifact correction (automatic identification of bad channels, Independent Component Analysis for ocular/muscle artifacts). It is critical to document each step for reproducibility: we leverage the MNE-BIDS-Pipeline [2] framework, which provides scripted execution of preprocessing steps with caching and provenance tracking. This ensures that the exact filtering, referencing (common average or linked1 [11] mastoids), and artifact-rejection parameters are recorded for audit and reuse. Recent work emphasizes [12] that even such preprocessing choices can dramatically affect downstream analyses, so consistency is vital in a clinical research context.

From the cleaned continuous EEG, we compute spectral power in canonical bands (delta 1–4 Hz, theta 4–8 Hz, alpha 8–13 Hz, beta 13–30 Hz, etc.) using Welch’s method or multitapering. Relative band power and ratios (e.g. theta/beta) are computed per channel and averaged over regions of interest. These qEEG features are saved in a structured format (CSV or JSON) along with metadata such as channel montages and patient demographics. This numeric summary forms the quantitative core of the report (for example, “Global theta power was elevated to 150% of the normative mean, consistent with prior findings in schizophrenia”). We also compute EEG complexity or connectivity metrics (entropy, coherence) as advanced optional features. Importantly, the feature extraction is coded in Python using open-source libraries (MNE, SciPy), and the entire pipeline from raw data to feature table can be re-run end-to-end, fulfilling reproducibility standards in bioengineering.

Clinical Data Integration

In addition to EEG, we integrate patient-specific clinical information to contextualize findings. For example, we include diagnosis, medication status, age, and standardized scores such as the BPRS (for psychiatric symptoms), HAM-D (for depression), or MoCA (for cognition). These data may come from an electronic health record or study database. In our framework, clinical scores are merged with EEG results so that the LLM can mention them (e.g. “The patient’s BPRS score was 28, indicating moderate [3] schizophrenia symptoms”). Prior studies often correlate EEG power changes with symptom scales. As one example, Newson & Thiagarajan note that schizophrenia severity was assessed using PANSS and BPRS in most EEG studies. By including such measures, the generated report can explain how EEG abnormalities align (or do not align) with clinical severity. This multimodal integration also allows RAG to retrieve literature linking EEG markers and clinical metrics. For instance, a search combining “theta power schizophrenia BPRS” may yield studies discussing EEG predictors of symptom improvement, which the narrative can cite.

Literature Retrieval and RAG-based Report Generation

To produce an evidence-based narrative, the system queries Europe PMC for relevant literature. Europe PMC is an open-access life sciences repository containing ~36 million article abstracts and 5 million full texts. We construct search queries using patient context (e.g. “schizophrenia EEG theta”), methodological terms (e.g. “qEEG spectrum analysis software”), and any novel findings (e.g. “delta power increase clinical meaning”). Using the Europe PMC RESTful API (or associated Python libraries), the pipeline retrieves top-ranking abstracts and open-access full-texts matching these queries. The selection is filtered for recency and relevance (for example, the last 10 years, human studies, English language).

The core of report generation uses a Retrieval-Augmented Generation model. Retrieved documents (titles, snippets, or passages) form an evidence bank. We then prompt a large language model (e.g. GPT-4 or a fine-tuned domain model) with both the structured patient/EEG data and key excerpts from the literature. The prompt instructs the LLM to write a structured report, ensuring each statement is grounded in the retrieved evidence. For example, in the LLM prompt we include: “Patient is a 30-year-old with schizophrenia (BPRS 30) whose EEG shows elevated theta power (mean 8 µV²). According to [Smith et al. 2022], increased theta power correlates with positive symptoms. Summarize these findings in a report with 9 references.” The RAG approach has demonstrated improved factual accuracy in medical summaries. Our application is analogous to systems like Alzheimer RAG, which fuse textual and imaging data 13 for case studies, and the EEG-MedRAG framework which builds hypergraphs of EEG knowledge and 6 patient data for causal diagnosis generation.

The output is a draft report comprising sections: Background (patient demographics, clinical history), EEG Acquisition (recording details, preprocessing), Results (qEEG features with normative comparisons), and Discussion (interpretation citing literature). In Discussion, the LLM references specific studies (e.g., “The observed theta increase aligns with reports of frontal slowing in schizophrenia 3”) and notes if findings contradict literature (e.g. no alpha slowing despite expectation). Each cited fact is traced to a reference from Europe PMC to maintain transparency. The language model is steered to a “report-writing” style, using sentence templates extracted from sample case studies.

Reproducibility and Open Pipeline Implementation

A key design goal is full reproducibility. All code is in Python and managed with version control. The data flow is modular: BIDS validation ensures input conformity, MNE/BIDS-Pipeline handles [1,11] preprocessing with cacheable steps, and feature computation scripts log their parameters. We containerize the environment (e.g. with Docker or Conda) so others can recreate the exact software setup. The RAG component is also documented: the retrieval queries and LLM prompts are saved alongside the results. This allows independent verification of the report content.

To facilitate reuse by researchers, we leverage community standards. Using BIDS format means that any 1 EEG dataset following BIDS-EEG can be plugged into the pipeline. We provide example Jupyter notebooks that walk through each step on sample data. The pipeline can run in parallel for multiple subjects, supporting large-scale studies (as advertised by the MNE-BIDS-Pipeline for hundreds of 11 datasets). Summaries of processing (filter logs, artifact rejection rates, feature distributions) are automatically compiled into a report PDF, enabling quick quality checks. By being open-source, this framework advances the ethos of reproducible neuroengineering practice.

Clinical Research Utility, Education, and Future Directions

This automated reporting tool has several utilities. In clinical research, it accelerates the generation of case studies and cohort summaries. Investigators can use it to standardize EEG report content across studies, reducing variability. The inclusion of RAG ensures that reports cite current literature, keeping interpretations up to date, which is crucial in fields where biomarker validity is evolving. In neuroengineering education, the pipeline serves as a teaching aid: students can explore how 12 preprocessing choices affect features, and how AI can assist in interpreting neurophysiological data.

The system can generate example cases for training, highlighting how spectral changes relate to diagnosis and literature. Looking forward, the framework can be extended. Future versions might incorporate other modalities (e.g. MRI or genetics) into the RAG context, enabling truly multimodal case reports. Improving the LLM’s domain specificity (through fine-tuning on neuroengineering literature) could reduce errors. There is also potential for real-time use: integrating with EEG acquisition software to update reports as data are collected. From a bioengineering perspective, such tools illustrate how AI can bridge raw data and clinical insight, embodying the translational promise of neuroinformatics.

Strengths, Limitations, and Outlook

This approach offers major strengths: automation greatly reduces expert time, promotes consistency, and ties findings to evidence. By using open pipelines and data standards, it encourages [8,5] reproducible research and democratizes complex EEG analysis for non-experts. However, limitations remain. EEG preprocessing is sensitive; suboptimal filtering or artifact correction can mislead analysis [12-15]. The quality of the LLM report hinges on retrieval: if relevant literature is missed or irrelevant documents are retrieved, the narrative may be skewed or incomplete. As noted in prior RAG studies, LLMs can still hallucinate or oversimplify in clinical contexts, so reports must be reviewed by experts. Data privacy is also a concern: patient data used in prompts should be de-identified and handled under appropriate governance.

In future work, evaluation is critical: we plan systematic testing of report accuracy by comparing AI generated reports with those by neurophysiologists. Advances in domain-specific LLMs and larger EEG text corpora will likely improve performance. Overall, merging automated EEG analytics with retrieval augmented AI represents a promising direction in bioengineering — one that could transform how we synthesize physiological data, clinical scores, and biomedical knowledge into actionable insights.

References

  1. Newson JJ, Thiagarajan TC (2019) EEG frequency bands in psychiatric disorders: a review of resting state studies. Front Hum Neurosci. 12: 521. [crossref]
  2. Kessler R, Enge A, Skeide MA (2025) How EEG preprocessing shapes decoding performance. Commun Biol 8: 1039.
  3. Wang Y, Luo H, Meng L (2025) EEG-MedRAG: Enhancing EEG-based clinical decision-making via hierarchical hypergraph retrieval-augmented generation. arXiv 2508: 13735.
  4. Masanneck L, Meuth SG, Pawlitzki M (2025) Evaluating base and retrieval-augmented LLMs with document or online support for evidence-based neurology. NPJ Digit Med 8: 137.
  5. Kuo SM, Tai SK, Lin HY, Chen RC (2025) Automated Clinical Trial Data Analysis and Report Generation by Integrating Retrieval Augmented Generation (RAG) and LLM Technologies. AI 6: 188.
  6. Gramfort A, Luessi M, Larson E, Engemann DA, Strohmeier D, et al. (2013) MEG and EEG data analysis with MNE-Python. Front Neurosci 7: 267. [crossref]
  7. EMBL-EBI Literature Services. Europe PMC database and text mining infrastructure (2025). MNE-BIDS-Pi pipeline https://mne.tools/mne-bids-pipeline/stable/
  8. How EEG preprocessing shapes decoding performance | Communications Biology
  9. https://www.nature.com/articles/s42003-025-08464-3?error=cookies_not_supported&code=3c2c65df-d6ec-42bf-b872- dd2b7e5a3d96
  10. EEG Frequency Bands in Psychiatric Disorders: https://pmc.ncbi.nlm.nih.gov/articles/PMC6333694/A
  11. Review of Resting State Studies – PMC Literature Services – Europe PMC database and text mining infrastructure https://www.ebi.ac.uk/about/teams/literature-services/
  12. Evaluating base and retrieval augmented LLMs with document or online support for evidence based neurology | npj Digital Medicine
  13. https://www.nature.com/articles/s41746-025-01536 y?error=cookies_not_supported&code=529c2d4a-150c-43b9-a98c-625f5a07fd01 [2508.13735]
  14. EEG-MedRAG: Enhancing EEG-based Clinical Decision-Making via Hierarchical Hypergraph Retrieval-Augmented Generation https://www.arxiv.org/abs/2508.13735
  15. Automated Clinical Trial Data Analysis and Report Generation by Integrating Retrieval. Augmented Generation (RAG) and Large Language Model (LLM) Technologies https://www.mdpi.com/2673-2688/6/8/188 AlzheimerRAG: Multimodal Retrieval Augmented Generation … – arXiv https://arxiv.org/html/2412.16701v1

A Commentary on “Better Treatment Outcomes with Aripiprazole Long-acting Injection in Community and Incarcerated Patients with Serious Mental Illness”

2 Replies

DOI: 10.31038/JDMR.2025813

 

Dear Editor,

Non-adherence to antipsychotic medication is a primary reason for treatment inefficacy in psychotic patients, particularly those with co-occurring substance use disorders (SUD) or alcohol use disorders (AUD). Poor adherence increases relapse risk, hospitalization rates, treatment costs, and the likelihood of legal issues, perpetuating a “revolving door” cycle of hospitalization and incarceration. In Greece, the impending implementation of Law 5129/2024, effective February 1, 2025, will integrate psychiatric services with the penitentiary system, prompting this study to assess aripiprazole LAI benefits for community and incarcerated patients in Eastern Crete. The findings aim to inform health policy, optimize resource allocation, and support psychiatric reform. As a third-generation antipsychotic, aripiprazole acts as a partial D2 and 5-HT1A agonist and 5-HT2A antagonist, approved by the FDA in 2002 [1]. Its unique mechanism [2] may reduce hyperdopaminergic activity in the mesolimbic system (antipsychotic effect) while enhancing hypodopaminergic activity in the prefrontal cortex, potentially alleviating negative symptoms and cognitive deficits [3-5]. Partial 5-HT1A agonism may also provide anxiolytic benefits [6]. Studies, including a 2015 trial in the UK and Canada, demonstrated aripiprazole’s ability to enhance dorsolateral prefrontal cortex (DLPFC) activation during working memory tasks, suggesting improved processing speed [7].

The study enrolled 55 patients: i) Community Patients (n=34): 70.6% male, mean age 42.3 ± 11.9 years, 44.1% with F20.0, 94% with Cluster C personality, 34.1% with psychoactive substance use, mean treatment duration 23.2 ± 18.3 months, ii) Incarcerated Patients (n=21): All male, mean age 37.6 ± 7.7 years, all with F29.0, 57.1% with Cluster B personality, 100% with psychoactive substance use, mean treatment duration 14.5 ± 11.3 months, iii) Significant differences were noted between groups in most demographic and medical history parameters, except for traumatic brain injury, mental retardation, age, and treatment duration.Participants were assessed using the WHOQOL-BREF and CGI-S scales, with outcomes compared pre- and post-aripiprazole LAI treatment over a minimum six-month period.

This study is the first in Greece to evaluate aripiprazole LAI for community patients with psychosis and SUD, and the first in Europe for incarcerated patients with unspecified psychosis and SUD. Observed outcomes may reflect temperamental traits, as noted by Favaretto et al. (2024) [8], which influence psychopathology severity and treatment engagement, especially in dual-diagnosis or Cluster B populations [9]. Our findings confirm significant improvements in quality of life, functionality, and hospitalization rates, aligning with Sampogna et al. (2023) on LAI benefits (10).Aripiprazole LAI also shows promise for AUD, with four community patients achieving abstinence and others reducing consumption. Animal studies support its efficacy in reducing ethanol-related behaviors [11,12]. Further research is needed to quantify cost savings and explore off-label use in AUD.

Ethical approval was granted by the General Hospital of Agios Nikolaos (decision 514/19-07-2023), Scientific Council of the General Hospital of Agios Nikolaos Lasithi of Crete – National Health System of Greece (Prot. No. 10/15-02-2023), 7th Sanitary Region of Crete (Prot. No. 28386/30-06-2023), and Ministry of Public Order (Prot. No. 10456/10-04-2023) under Law 4812/2021, Article 87. The studies were conducted in accordance with the local legislation and institutional requirements.

Based on: 1) Our study under comment, 2) our clinical experience and at the same time on the data of a larger sample that we have already collected and are studying in recent months, we have been led to the conclusion that it would be extremely interesting for our research to study the therapeutic stabilization of our patients in the first three months of treatment with aripiprazole LAI. The recent results to which our constantly evolving research has led us have given us all the important indications of an effective and at the same time faster response to therapy, which could occur before 6 months from the first day of administration of the medication.

Evaluating the results of our studies [13-15] and guided by our clinical examination based on our medical skill and experience, we observe a gradual improvement in the clinical status of patients (patients in community and incarcerated patients) after the first two months of administration of the Depot antipsychotic therapy, such as their daily functionality, their affective and cognitive response to any form of everyday stimuli [16,17], in combination with the improvement of their quality of life (as evidenced by the systematic medical clinical examination and medical observation of patients, by the management of the crisis in their daily life and by the interactive relationship of the patients with familiar persons in different environments) and the reduction of their hospitalizations due to the hard core of their disease. All these lead us to think about the possibility of having better clinical outcomes and satisfactory indication of stabilization from the treatment even in the first three months.

Such a possibility would provide to patients faster relief from the symptoms of their illness and would reduce more quickly the possibility of dangerous exacerbations of the disease, which are associated with the triggering of inflammatory processes in the body and the sensitization of the immune system. Moreover, such a possibility would reduce the possible threat of the patients’ integrity and the integrity of the people around them from clinical manifestations of inappropriate behaviors of the patients that characterize the disease (for example, positive symptoms of the disease). Finally, would encourage the compliance of the patients in the faithful adherence and continuation of their treatment and would restore the general pathogenesis created in their environment by the effects of their illness.

References

  1. Di Sciascio G, Riva MA (2015) Aripiprazole: from pharmacological profile to clinical Neuropsychiatr Dis Treat 11: 2635.
  2. Abilify 5 mg/ml solution for injection (intramuscular) – summary of product characteristics (SmPC)
  3. DeLeon A, Patel NC, Crismon ML (2004) Aripiprazole: a comprehensive review of its pharmacology, clinical efficacy, and Clin Ther 26: 649-66.
  4. Millan MJ (2003) The neurobiology and control of anxious states. Prog Neurobiol 70: 83-244.
  5. Murphy A, Dursun S, McKie S, Elliott R, Deakin JF (2016) An investigation into aripiprazole’s partial D2 agonist effects within the dorsolateral prefrontal cortex during working memory in healthy volunteers. Psychopharmacol (Berl) 233: 1415-26.
  6. Lee JS, Lee JD, ParkH-J, Oh M-K, Chun JW, et (2013) Is the GABA system related to the social competence improvement effect of aripiprazole? An (18) F-fluoroflumazenil PET study. Psychiatry Investig 10: 75-80.
  7. Hahn M, Roll SC (2016) Dosing recommendations of aripiprazole depot with strong cytochrome P450 3A4 inhibitors: A relapse risk. Drug Saf Case Rep 3: 5.
  8. Favaretto E, Bedani F, Brancati GE, De Berardis D, Giovannini S, et (2024) Synthesising 30 years of clinical experience and scientific insight on affective temperaments in psychiatric disorders: State of the art. J Affect Disord 362: 406-415.
  9. Burnette EM, Nieto SJ, Grodin EN, Meredith LR, Hurley B, et (2022) Novel agents for the pharmacological treatment of alcohol use disorder. Drugs 82: 251-74.
  10. Sampogna G, Di Vincenzo M, Giuliani L, Menculini G, Mancuso E, et al. (2023) A systematic review on the effectiveness of antipsychotic drugs on the quality of life of patients with Brain Sci 13: 1577.
  11. Ingman K, Kupila J, Hyytia P, et (2006) Effects of aripiprazole on alcohol intake in an animal model of high-alcohol drinking. Alcohol 41: 391-8.
  12. Jerlhag E (2008) The antipsychotic aripiprazole antagonizes the ethanol- and amphetamine-induced locomotor stimulation in Alcohol 42: 123-7.
  13. Koiliari EI, Mouzas I, Alevizopoulos G, Lesch O, Walter H, et (2024) Long-acting injectable aripiprazole in patients with psychosis is associated with improved quality of life, better general clinical outcome and fewer hospitalizations. European Psychiatry 67: S729-S729.
  14. Pasparakis EL, Mouzas I, Detorakis GI, Koiliari EI (2025) Analysis of the therapeutic effect of the long-acting injectable form of aripiprazole in incarcerated adult males in European Psychiatry 68: S385-S385.
  15. Koiliari EI, Mouzas I, Detorakis GI, Pasparakis EL (2025) The use of depot antipsychotics in patients with dual diagnosis of psychosis and substance use disorder is associated with improved quality of life, better general clinical outcome and fewer European Psychiatry 68: S429-S430.
  16. Koiliari E, Roussos P, Pasparakis E, Lencz T, Malhotra A, et (2014) The CSMD1 genome-wide associated schizophrenia risk variant rs10503253 affects general cognitive ability and executive function in healthy males. Schizophr Res 154: 42-7.
  17. Pasparakis E, Koiliari E, Zouraraki C, Tsapakis EM, Roussos P, et (2015) The effects of the CACNA1C rs1006737 A/G on affective startle modulation in healthy males. Eur Psychiatry 30: 492-8.

Design, Synthesis, and Anticancer Activity of 1-(2-(Adamantan-1-yl)-1H-indol-5-yl)-3-Substituted Thiourea Derivatives as CDK9 Inhibitors

3 Replies

DOI: 10.31038/JPPR.2025814

Abstract

Objective: A novel Cdk9 inhibitor 1-(2-adamantane-1-yl-1H indole-5-yl)-3-substituted thiourea derivative was designed and synthesized and their anti-gastric cancer activities were studied.

Methods: A series of target compounds 7a-7m were synthesized from amantadane formyl chloride by 6-step reactions. The structures of the target compounds were identified by 1H NMR, 13C NMR and HRMS. MTT assay was used to detect the inhibitory effect of synthetic compounds on the growth of gastric cancer cells, and Western blot assay was used to detect the regulatory effect of hit compound on downstream signaling pathways. Results: The results showed that the target compounds had certain inhibitory activity on the growth of gastric cancer cells, among which compound 7l had the best activity on the gastric cancer cell line (SGC-7901) with IC50 value of 2.26 ± 0.04 μM, and 7I had little toxicity on normal gastric epithelial cells (IC50 > 100 μM). In addition, 7I can bind to CDK9 and inhibit pSer2 expression in gastric cancer cells in a concentration dependent manner. Finally, molecular docking study showed that 7l can stably bind to the active site of CDK9 and has a high binding affinity.

Conclusion: This series of compounds has good anti-gastric cancer activity and has the significance of further study.

Keywords

Adamantane derivatives; Indole; Gastric cancer; CDK9

Gastric cancer is one of the most common cancers worldwide and ranks as the third leading cause of cancer-related deaths [1]. Middle-aged and elderly individuals are at high risk, and gastric cancer is often diagnosed at an advanced stage, with a five-year survival rate of less than 30% [2,3]. Therefore, understanding the occurrence and progression of gastric cancer, along with the potential discovery of novel diagnostic and prognostic biomarkers, is crucial for improving clinical outcomes. Clinically, the primary treatment for gastric cancer is surgical intervention, supplemented by pharmacological therapy [4]. Although chemotherapy can extend the survival of patients to some extent, conventional chemotherapeutic agents suffer from poor specificity, significant toxicity, side effects, and a tendency to induce drug resistance, all of which adversely affect treatment efficacy and patient survival [5]. Thus, identifying effective drugs with favorable therapeutic profiles for gastric cancer is of great importance. Cyclin-dependent kinases (CDKs) are a family of serine/threonine protein kinases that play vital roles in cell cycle progression and transcriptional regulation [6-9]. Recent studies have shown that CDKs are overexpressed in various cancers, leading to uncontrolled cell proliferation and drug resistance [10]. CDK9, a key member of the CDK family, is essential for stable RNA transcriptional elongation. CDK9 and cyclin T form the positive transcription elongation factor b (P-TEFb) complex, which promotes transcriptional elongation through phosphorylation of RNA polymerase II (RNAPII) [11,12]. Recent evidence indicates that CDK9 plays a critical role in numerous human cancers, including cervical, prostate, and lung cancers [13-18]. Dual-luciferase reporter assays have demonstrated that miR-613 inhibits the metastasis and progression of gastric cancer cells by downregulating CDK9 gene expression [19]. Therefore, targeting CDK9 to interfere with the development and progression of gastric cancer holds therapeutic promise. Building on previous work, this study designed and synthesized a novel series of CDK9 inhibitors (Figure 1). The influence of different substituted R groups on the antitumor activity of these thiourea compounds was investigated to identify more potent 1-(2-(adamantan-1-yl)-1H-indol-5-yl)-3-substituted thiourea derivatives. The synthetic route for target compounds 7a–7l is illustrated in Figure 1.

Figure 1: Synthetic route of compounds 7a～7l.

Materials and Methods

Chemicals and Reagents

Starting materials were purchased from Shanghai Titan Scientific Co., Ltd. All reagents were of analytical grade and used without further purification. Solvents were used as received. Thin-layer chromatography (TLC) silica gel (Qingdao Marine Chemical Factory, 60–100 mesh) and MTT powder (ST1537, Beyotime) were used.

Instruments

NMR spectra were recorded on a Bruker Avance 600 spectrometer using DMSO-d6 or CDCl3 as the solvent with TMS as the internal standard. Mass spectra were obtained using an Agilent 6230 mass spectrometer. A Thermo microplate reader was used for absorbance measurements.

Methods

MTT Assay for Cell Viability

The MTT assay was used to assess cell survival and growth. Cells were digested and seeded into 96-well plates at a density of 3,000 cells per well. After cell attachment, they were treated with the respective compounds or solvent, with cisplatin used as a positive control. After 48 hours of incubation, the medium was replaced with fresh medium containing MTT solution (1.2 mg/mL), and the cells were incubated for another 3 hours. The MTT formazan product was dissolved in DMSO, and the absorbance was measured at 492 nm using a microplate reader.

CDK9 Inhibition Assay

The ADP-Glo Kinase Assay was employed to determine the inhibitory activity of the compounds against CDK9 protein, conducted by Innovative CRO + Explorer (Beijing, China). Kinase activity was assessed in a buffer containing 50 mM Hepes, 10 mM MgCl2, 0.01% Brij 35, 1 mM EGTA, 2 mM DTT, and ddH2O. Test compounds were prepared in DMSO. The inhibition rate against CDK9/cyclin T1 (ATP: 20 μM) was tested for all compounds at a concentration of 1 μM.

Molecular Docking

Molecular docking was performed to explore the interaction mode between compound 7l and CDK9. The crystal structure of CDK9/cyclin T1 (PDB ID: 7NWK, resolution: 2.81 Å) was downloaded from the Protein Data Bank (https://www.rcsb.org/structure/7NWK). The protein structure was prepared using the Protein Preparation Wizard module in Schrödinger software (V 2023-1) under default parameters, including removal of water and solvent molecules, addition of hydrogen atoms, assignment of charges, completion of missing amino acid residues, optimization of the hydrogen bond network, and energy minimization using the OPLS4 force field. The structure of 7l was prepared using LigPrep (Schrödinger, LLC, New York, NY, 2023) under the OPLS4 force field with default parameters, including hydrogen addition, charge assignment, and generation of possible protonation states at pH 7.0 ± 2.0. Molecular docking was performed using the Ligand Docking module in Schrödinger software, docking 7l into the ATP-binding site of CDK9. The docking box was centered on the centroid of the original crystal ligand, and extra precision (XP) docking was employed. The binding energy of the 7l-CDK9 complex was calculated using the MM-GBSA module. Maestro (Version 12.5, Schrödinger) was used for visualization, and PyMOL (Version 2.5.4, Schrödinger, LLC) was used for rendering and presentation.

Experimental Methods and Results

Synthesis of Intermediate N-(o-Tolyl)adamantane-1-Carboxamide (2)

In a dried 50 mL round-bottom flask, toluene (10 mL), adamantane-1-carbonyl chloride (0.99 g, 5 mmol), o-toluidine (0.54 g, 5 mmol), and potassium carbonate (0.69 g, 5 mmol) were added successively. The mixture was stirred and heated to 80°C, and the reaction progress was monitored by TLC. After completion, the reaction mixture was cooled, filtered, washed with water, and dried to afford a crude white solid, which was recrystallized from ethanol to yield N-(o-tolyl)adamantane-1-carboxamide (1.17 g, 87%). 1H-NMR(600 MHz, CDCl3) δ: 7.88 (d, J=8.1 Hz, 1H), 7.24-7.18 (m, 2H), 7.17 (d, J=7.5Hz, 1H), 7.05 (dt, J=1.1, 7.4 Hz, 1H), 2.26 (s, 3H), 2.11 (brs, 3H), 1.99 (d, J=2.6 Hz, 6H), 1.77 (m, 6H).

Synthesis of Intermediate 2-(Adamantan-1-yl)-1H-indole (3)

A dried 25 mL three-necked flask was charged with N-(o-tolyl)adamantane-1 carboxamide (0.285 g, 1.0 mmol) and THF (15 mL). The mixture was cooled to 0–5°C under nitrogen, and n-butyllithium (2 mL) was added dropwise. The reaction was monitored by TLC. Upon completion, the pH was adjusted to 7.0–7.5 with 5% dilute hydrochloric acid. The mixture was extracted, and the organic phase was dried over anhydrous Na2SO4, filtered, and concentrated under reduced pressure. The crude product was purified by column chromatography (eluent: petroleum ether/ethyl acetate=10: 1, v/v) to yield 2-(adamantan-1-yl)-1H-indole (0.166 g, 66.1%). 1H-NMR (600 MHz, CDCl3) δ: 7.30 (d, J=7.9 Hz, 1H), 7.18 (dd, J=0.5, 8.1 Hz, 1H), 6.88 (dt, J=1.1, 7.5 Hz, 1H), 6.83-6.77 (m, 1H), 5.98 (d, J=0.6 Hz, 1H), 1.97 (d, J=2.6 Hz, 3H), 1.93 (d, J=2.9 Hz, 6H), 1.73 (brs, 6H).

Synthesis of Intermediate 2-(Adamantan-1-yl)-5-nitro-1H-indole (4)

A 25 mL three-necked flask was charged with 2-(adamantan-1-yl)-1H-indole (0.25 g, 1.0 mmol) and concentrated sulfuric acid (0.8 mL). A solution of sodium nitrate (0.085 g, 1.0 mmol) in sulfuric acid (0.8 mL) was added dropwise at 0°C. After addition, the reaction was maintained for 1–2 hours and monitored by TLC. Upon completion, a large amount of ice water was added, and a yellow solid precipitated. The solid was filtered, washed with water, and dried. The crude product was purified by column chromatography (eluent: petroleum ether/ethyl acetate=5: 1, v/v) to yield 2 (adamantan-1-yl)-5-nitro-1H-indole (0.24 g, 82.8%) as a yellow solid. 1H-NMR (600 MHz, DMSO-d6) δ: 11.65 (brs, 1H), 8.44 (d, J=2.2 Hz, 1H), 7.93 (dd, J=2.2 ,9.0 Hz, 1H), 7.44 (d, J=8.8 Hz, 1H), 6.38 (d, J=1.5 Hz, 1H), 2.07 (brs,3H), 1.98 (brs, 6H), 1.81-1.67 (m, 6H);13C-NMR (150 MHz, DMSO-d6) δ: 154.0, 140.8, 139.9, 127.7, 116.9, 116.3, 111.4, 98.2, 41.9, 36.6, 34.1, 28.2; HRMS (ESI) m/z: 297.1599 [M+H]+, Calt: 297.1598 [M+H]+.

Synthesis of Intermediate 5-Amino-2-(adamantan-1-yl)-1H-indole (5)

In a 250 mL three-necked flask, ethanol (100 mL), acetic acid (10 mL), water (20 mL), and iron powder (8.62 g, 0.15 mol) were added. The mixture was heated to 75°C, and 2-(adamantan-1-yl)-5-nitro-1H-indole (11.4 g, 0.038 mol) was added in portions. The reaction was maintained for 2 hours and monitored by TLC. After completion, the reaction mixture was hot-filtered, and the filtrate was concentrated under reduced pressure to remove the solvent. Water (80 mL) was added, followed by ethyl acetate (80 mL) with stirring. The pH was adjusted to 7–8 with sodium bicarbonate. The organic phase was separated, dried over anhydrous Na22SO44, filtered, and purified by column chromatography (eluent: petroleum ether/ethyl acetate=2: 1, v/v) to yield 5 amino-2-(adamantan-1-yl)-1H-indole (6.9 g, 67.0%) as a red solid. 1H-NMR (600 MHz, CDCl3) δ: 7.80 (brs, 1H), 7.10 (d, J=8.4 Hz, 1H), 6.86 (d, J=2.2 Hz, 1H), 6.57 (dd, J=2.2, 8.4 Hz, 1H), 6.05 (dd, J=0.7, 2.2 Hz, 1H), 3.23-3.65 (m, 2H), 2.09 (brs, 3H), 1.96 (d, J=2.38 Hz, 6H), 1.78 (q, J=12.2 Hz, 6H);13C-NMR (150 MHz, CDCl3) δ: 149.9,139.3, 130.3, 129.4, 111.6, 110.8, 105.3, 95.5, 42.6, 36.8, 33.7, 28.5; HRMS (ESI) m/z: 267.1858 [M+H]+, Calt: 267.1856 [M+H]+.

Synthesis of Intermediate 5-Isothiocyanato-2-(adamantan-1-yl)-1H-indole (6)

In a dried 100 mL three-necked flask, a solution of 5-amino-2-(adamantan-1-yl)-1H indole (4.10 g, 15.5 mmol) in toluene (20 mL) and triethylamine (3 mL) was added. Carbon disulfide (3.50 g, 46.5 mmol) was added dropwise slowly. The reaction was stirred at room temperature for 8 hours. The mixture was filtered and dried, then dissolved in dichloromethane (30 mL). Triphosgene (5.0 g, 17 mmol) was added dropwise at 0–5°C. After 2 hours, the reaction was monitored by TLC. Upon completion, the product was purified by column chromatography (eluent: petroleum ether/ethyl acetate=10: 1, v/v) to yield 5-isothiocyanato-2-(adamantan-1-yl)-1H-indole (2.89 g, 64.0%) as a white solid. 1H-NMR(600 MHz, CDCl3) δ: 7.40 (d, J=1.8 Hz, 1H ), 7.23 (d, J=8.4 Hz, 1H, ), 6.98 (dd, J=2.0, 8.4 Hz, 1H), 6.20 (d, J=1.5 Hz, 1H ), 2.14 2.08 (m, 3H), 1.99-1.94 (m, 6H), 1.85-1.74 (m, 6H); 13 C-NMR (150 MHz, CDCl3) δ: 151.4, 134.1, 131.8, 128.7, 122.6, 119.0, 117.3, 111.2, 96.8, 42.5, 36.7, 33.9, 28.4; HRMS (ESI) m/z: 309.1422 [M+H]+, Calt: 309.1420 [M+H]+.

Synthesis of 1-(2-(Adamantan-1-yl)-1H-indol-5-yl)-3-Substituted Thiourea Derivatives (7a–7l)

In a 25 mL reaction flask, 5-isothiocyanato-2-(adamantan-1-yl)-1H-indole (0.145 g, 0.5 mmol) and toluene (10 mL) were added. The appropriate amine (0.5 mmol) was added, and the mixture was heated to 80°C. The reaction was monitored by TLC. After cooling, the mixture was filtered and dried to afford the target compounds as white solids in yields of 70.0–88.6%.

7a: white solid, yield 70.1%, mp 198～200℃, 1H NMR (400 MHz, DMSO-d6) δ: 10.92 (s, 1H), 9.25 (brs, 1H), 7.27 (dd, J=3.2, 4.95 Hz, 2H), 6.83 (dd, J=1.7 8.5 Hz, 1H), 6.08 (d, J=1.71 Hz, 1H), 3.47-3.40 (m, 2H), 2.06 (brs, 3H), 1.96 (d, J=1.96 Hz, 6H), 1.76 (br, 6H), 1.54-1.41 (m, 2H), 1.32-1.19 (m, 6H), 0.87 (t, J=6.79 Hz, 3H);13C NMR (100 MHz, DMSO-d6): δ 181.0, 152.5, 151.1, 135.3, 134.6, 128.4, 116.8, 111.5, 95.8, 44.5, 42.2, 36.8, 33.9, 31.5, 29.1, 28.3, 26.5, 22.5, 14.4; HRMS (ESI) m/z: 410.2625 [M+H]+, Calt: 410.2624 [M+H]+.

7b: white solid, yield 78.4%, mp 201～203℃, 1H NMR (400 MHz, DMSO-d6) δ: 10.90 (s, 1H), 9.21 (s, 1H), 7.32 (s, 1H), 7.25 (d, J=8.44 Hz, 1H), 6.96 (brs, 1H), 6.87 (dd, J=1.7, 8.4 Hz, 1H), 6.07 (d, J=1.3 Hz, 1H), 2.06 (brs, 3H), 1.96 (brs, 6H), 1.89-1.83 (m, 2H), 1.80-1.71 (m, 6H), 1.64 (d, J=12.6 Hz, 2H), 1.54 (d, J=12.7 Hz, 1H), 1.32-1.04 (m, 5H); 13C NMR (100 MHz, DMSO-d6) δ: 180.0, 151.0, 134.4, 130.1, 128.3, 118.8, 116.4, 111.3, 95.7, 52.9, 42.2, 36.8, 33.9, 32.4, 28.4, 25.6, 25.1; HRMS (ESI) m/z: 408.2469 [M+H]+, Calt: 408.2468 [M+H]+.

7c: white solid, yield 80.1%, mp 221～223℃, 1H NMR (400 MHz, DMSO-d6) δ: 10.91 (s, 1H), 9.66 (s, 1H), 9.35 (brs, 1H), 7.50 (d, J=7.8 Hz, 2H), 7.42 (s, 1H), 7.33-7.25 (m, 3H), 7.13-7.07 (m, 1H), 6.98 (dd, J=1.8, 8.5Hz, 1H), 6.10 (d, J=1.6Hz, 1H), 2.07 (brs, 3H), 1.97 (s, 6H), 1.84-1.66 (m, 6H); 13C NMR (100 MHz, DMSO-d6) δ: 180.4, 151.0, 140.3, 134.5, 130.5, 128.7, 128.3, 124.6, 124.3, 119.0, 116.6, 111.1, 95.8, 42.3, 36.8, 33.9, 28.4; HRMS (ESI) m/z: 402.1999 [M+H]+, Calt: 402.1998 [M+H]+.

7d: white solid, yield 78.8%, mp 225～226℃; 1H NMR (400 MHz, DMSO-d6) δ: 10.91 (s, 1H), 9.54 (s, 1H), 8.87 (brs, 1H), 7.42 (d, J=1.5 Hz, 1H), 7.30-7.24 (m, 2H), 7.22 (dd, J=1.5, 6.9 Hz, 1H), 7.19-7.09 (m, 2H), 6.98 (dd, J=1.9, 8.5 Hz, 1H), 6.10 (d, J=1.7 Hz, 1H), 2.24 (s, 3H), 2.07 (brs, 3H), 1.97 (d, J=2.0 Hz, 6H), 1.82 1.72 (m, 6H); 13C NMR (100 MHz, DMSO-d6) δ: 181.1, 151.0, 138.8, 135.3, 134.6, 130.6, 128.7, 128.3, 126.6, 126.3, 119.2, 116.9, 111.2, 95.8, 42.3, 36.8, 33.9, 28.4, 18.4;HRMS (ESI) m/z: 416.2152 [M+H]+,Calt: 416.2155 [M+H]+.

7e: white solid, yield 75.6%, mp 234～236℃;1H NMR (400 MHz, DMSO-d6) δ: 10.91 (s, 1H), 9.62 (s, 1H), 9.27 (brs, 1H), 7.41 (s, 1H), 7.32-7.24 (m, 3H), 7.19 (t, J=8.1 Hz, 1H), 6.97 (dd, J=1.8, 8.5 Hz, 1H), 6.92 (d, J=7.5 Hz, 1H), 6.09 (d, J=1.5 Hz, 1H), 2.28 (s, 3H), 2.07 (brs, 3H), 1.97 (brs, 6H), 1.83-1.63 (m, 6H);13C NMR (100 MHz, DMSO-d6) δ: 180.3, 151.0, 140.2, 137.9, 134.5, 130.5, 128.5, 128.2, 125.3, 124.9, 121.5, 119.1, 116.6, 111.1, 95.8, 42.3, 36.8, 33.9, 28.4, 21.5; HRMS (ESI) m/z: 416.2150 [M+H]+,Calt: 416.2155 [M+H]+.

7f: white solid, yield 79.5%, mp 227～228℃; 1H NMR (600 MHz, DMSO-d6) δ: 10.92 (brs, 1H), 9.59 (brs, 1H), 9.26 (brs, 1H), 7.41 (s, 1H), 7.35 (d, J=8.0 Hz, 2H), 7.27 (d, J=8.4 Hz, 1H), 7.11 (d, J=8.0 Hz, 2H), 6.97 (d, J=7.7 Hz, 1H), 6.09 (s, 1H), 2.28 (s, 3H), 2.07 (brs, 3H), 1.97 (brs, 6H), 1.81-1.70 (m, 6H); 13C NMR (150 MHz, DMSO-d6) δ: 180.4, 151.0, 137.7, 134.5, 133.8, 130.5, 129.1, 128.2, 124.6, 119.1, 116.6, 111.1, 95.7, 42.3, 36.8, 33.9, 28.4, 21.0;HRMS (ESI) m/z: 416.2151 [M+H]+,Calt: 416.2155 [M+H]+.

7g: white solid, yields 80.1%, mp 244～246℃; 1H NMR (400 MHz, DMSO-d6) δ: 10.90 (s, 1H), 9.50 (s, 1H), 9.14 (brs, 1H), 7.41 (d, J=0.9 Hz, 1H), 7.33 (d, J=8.9 Hz, 2H), 7.27 (d, J=8.4 Hz, 1H), 6.97 (dd, J=1.8, 8.5 Hz, 1H), 6.88 (d, J=8.9 Hz, 2H), 6.09 (d, J=1.5 Hz, 1H), 3.74 (s, 3H), 2.07 (brs, 3H), 1.97 (brs, 6H), 1.82-1.71 (m, 6H);13C NMR (100 MHz, DMSO-d6) δ: 180.7, 156.8, 151.0, 134.5, 133.1, 130.5, 128.3, 126.7, 119.1, 116.7, 113.9, 111.1, 95.7, 55.7, 42.3, 36.8, 33.9, 28.4;HRMS (ESI) m/z: 432.2106 [M+H]+,Calt: 432.2104 [M+H]+.

7h: white solid, yields 73.1%, mp 208～210℃; 1H NMR (600 MHz, DMSO-d6) δ: 10.93 (s, 1H), 9.70 (brs, 1H), 9.31 (brs, 1H), 7.46 (dd, J=5.1, 8.6 Hz, 2H), 7.40 (s, 1H), 7.27 (d, J=8.6 Hz, 1H), 7.14 (t, J=8.8 Hz, 2H), 6.96 (dd, J=1.4, 8.5 Hz, 1H), 6.09 (d, J=1.5 Hz, 1H), 2.07 (brs, 3H), 1.97 (brs, 6H), 1.80-1.72 (m, 6H); 13C NMR (150 MHz, DMSO-d6) δ: 180.7, 159.5 (d, J=238.5 Hz), 151.0, 136.7, 134.6, 130.3, 128.3, 126.9, 119.0, 116.6, 115.2(d, J=21.0 Hz), 111.2, 95.8, 42.2, 36.8, 33.9, 28.4; (ESI) m/z: 420.1904 [M+H]+,Calt: 420.1904 [M+H]+.

7i: white solid, yields 79.1%, mp 245～246℃; 1H NMR (600 MHz, DMSO-d6) δ: 10.94 (brs, 1H), 9.78 (brs, 1H), 9.44 (brs, 1H), 7.52 (d, J=8.4 Hz, 2H), 7.41 (brs, 1H), 7.35 (d, J=8.8 Hz, 2H), 7.27 (d, J=8.4 Hz, 1H), 6.96 (d, J=8.0 Hz, 1H), 6.09 (s, 1H), 2.07 (brs, 3H), 1.97 (brs, 6H), 1.81-1.72 (m, 6H); 13C NMR (150 MHz, DMSO d6) δ: 180.4, 151.1, 139.4, 134.6, 128.7, 128.4, 128.2, 126.0, 125.8, 119.0, 116.6, 111.2, 95.8, 42.2, 36.8, 33.9, 28.4; (ESI) m/z: 436.1605 [M+H]+,Calt: 436.1609 [M+H]+.

7j: white solid, yield 82.3%, mp 218～220℃; 1H NMR (400 MHz, DMSO-d6) δ: 10.93 (s, 1H), 9.95 (s, 1H), 9.71 (brs, 1H), 7.78 (d, J=8.4Hz, 2H), 7.65 (d, J=8.6 Hz, 2H), 7.43 (s, 1H), 7.28 (d, J=8.4 Hz, 1H), 6.99 (dd, J=1.7, 8.5 Hz, 1H), 6.10 (d, J=1.5Hz, 1H), 2.07 (brs, 3H), 1.97 (d, J=2.0 Hz, 6H), 1.82-1.69 (m, 6H);13C NMR (100 MHz, DMSO-d6) δ: 180.3, 151.1, 144.3, 134.6, 130.3, 128.3, 125.7, 123.5, 118.8, 116.5, 111.2, 95.8, 42.2, 36.8, 33.9, 28.4; (ESI) m/z: 470.1872 [M+H]+,Calt: 470.1872 [M+H]+.

7k: white solid, yield: 88.6%; mp 242-243℃; 1H NMR (600 MHz, DMSO-d6) δ: 10.78-10.94 (m, 1H), 9.42 (brs, 1H), 8.77 (brs, 1H), 7.40 (s, 1H), 7.26 (d, J=8.4 Hz, 1H), 7.10 (d, J=7.8 Hz, 1H), 7.01 (s, 1H), 6.98-6.92 (m, 2H), 6.14-5.94 (m, 1H), 2.25 (s, 3H), 2.19 (s, 3H), 2.05 (brs, 3H), 1.96 (brs, 6H), 1.82-1.70 (m, 6H);13C NMR (150 MHz, DMSO-d6) δ: 151.0, 135.8, 135.1, 134.6, 131.2, 129.4, 128.7, 128.6, 128.3, 126.9, 119.2, 116.9, 95.8, 42.3, 36.8, 33.9, 28.4, 21.0, 18.3;(ESI) m/z: 430.2310 [M+H]+,Calt: 430.2311 [M+H]+.

7l: white solid, yield 72.8%, mp 228～230℃;1H NMR (400 MHz, DMSO-d6) δ: 10.94 (s, 1H), 9.85 (brs, 1H), 8.96 (brs, 1H), 7.52 (d, J=6.7 Hz, 1H), 7.42 (s, 1H), 7.31-7.23 (m, 2H), 7.07-7.01 (m, 1H), 6.97 (dd, J=1.8, 8.5Hz, 1H), 6.11 (d, J=1.6 Hz, 1H), 2.07 (brs, 3H), 1.97 (d, J=2.2 Hz, 6H), 1.83-1.69 (m, 6H);13C NMR (100 MHz, DMSO-d6) δ: 181.6, 161.7, 156.2, 151.1 (d, J=25.0 Hz), 134.7, 131.0, 130.2 (d, J=25.0 Hz), 129.4, 128.7, 128.3, 124.9, 119.0, 116.8, 111.3, 111.1, 104.5, 95.8, 42.2, 36.8, 33.9, 28.4; (ESI) m/z: 438.1812 [M+H]+,Calt: 438.1810 [M+H]+.

Results and Discussion

Structure-Activity Relationship (SAR) Discussion

The target compounds were synthesized starting from adamantane-1-carbonyl chloride and o-toluidine to yield N-(o-tolyl)adamantane-1-carboxamide (compound 2), which was then reacted with n-butyllithium to give 2-(adamantan-1-yl)-1H-indole (compound 3). Nitration of 3 afforded 2-(adamantan-1-yl)-5-nitro-1H-indole (compound 4), which was reduced to 5-amino-2-(adamantan-1-yl)-1H-indole (compound 5). Treatment of 5 with triphosgene yielded 5-isothiocyanato-2 (adamantan-1-yl)-1H-indole (compound 6). Reaction of 6 with various amines gave the target compounds 7a–7l, whose structures were confirmed by 1H NMR, 13C NMR, and HRMS. The in vitro inhibitory activities of the synthesized compounds against the gastric cancer cell line SGC-7901 were evaluated using the MTT assay, and the results are summarized in Table 1. The effects of different R groups (alkyl, cycloalkyl, substituted aryl, and fused aromatic rings) on the inhibitory activity against SGC-7901 were investigated. According to Table 1, the order of potency for R=hexyl, cyclohexyl, phenyl, and naphthyl was 7c (phenyl) > 7b (cyclohexyl) > 7a (hexyl). For monosubstituted phenyl derivatives (7d–7j), the order was 4-Cl (7i) > 4-F (7h) > 4-OCH3 (7g) > 4-CF3 (7j) > 2-CH3 (7d) > 3-CH3 (7e) > 4-CH3 (7f). For disubstituted phenyl derivatives (7k–7l), 2,4-diCl (7l) > 2,4-diCH3 (7k). Compound 7l exhibited the most potent activity, with an IC50 value of 2.26 ± 0.04 μM. Moreover, 7l showed low toxicity toward normal gastric epithelial cells (IC50>100 μM).

Table 1: Structure, kinase and cell inhibitory activity of the targeted compounds.

Analysis of the Binding Mode of 7l with CDK9

Molecular docking studies were performed to predict the binding mode of 7l with Compound Structure SGC-7901 (IC50/μM) CDK9 inhibiton (@ 1μM) R 7a 15.35 ± 0.23 50.1% 7c 10.11 ± 0.12 58.1% 7e 14.56 ± 0.20 52.3% 7g O 8.45 ± 0.07 73.8% 7i Cl 7.35 ± 0.11 79.5% 7k 13.18 ± 0.47 54.1% 7m 24.16 ± 0.14 45.9% 7b 21.24 ± 0.34 40.2% 7d 12.10 ± 0.45 54.3% 7f 17.23 ± 0.11 49.7% 7h F 8.09 ± 0.37 75.5% 7j CF3 9.76 ± 0.20 60.1% 7l F F 2.26 ± 0.04 85.6% CDK9. The results indicated that 7l binds within the ATP-binding pocket of CDK9, and its three-dimensional structure fits well into the active site (Figure 2A). As shown in Figure 2B, the indole core and thiourea moiety of 7l form stable interactions with key residues of CDK9. The hydrogen atom on the indole nitrogen acts as a hydrogen bond donor, forming a strong hydrogen bond (1.80 Å) with the carbonyl oxygen of GLU107 in the hinge region, indicating that the core of 7l is well-anchored in the hinge region of the CDK9 active site. Additionally, the hydrogen atoms on the thiourea moiety provide further opportunities for interactions with amino acid residues in the active site. The docking results showed that a hydrogen atom on the thiourea group acts as a hydrogen bond donor, forming a hydrogen bond (2.20 Å) with GLY28 of CDK9. Furthermore, the binding free energy of 7l with CDK9, calculated using the MM-GBSA method, was –32.86 kcal/mol, further confirming the high binding affinity of 7l for CDK9.

Through a molecular hybridization strategy, twelve new 1-(2-(adamantan-1-yl) 1H-indol-5-yl)-3-substituted thiourea derivatives (7a–7l) were synthesized and characterized by 1H NMR, 13C NMR, and HRMS. The MTT and CDK9 inhibition assay demonstrated that these compounds inhibit the growth of gastric cancer cells, with compound 7l exhibiting the most potent activity. Molecular docking studies indicated that 7l binds stably to the active site of CDK9 with high binding affinity. Future work will focus on further evaluation of 7l.

Figure 2: Binding pattern of compound 7l to CDK9. A. Schematic representation of the surface of 7l binding to CDK9; B. The three-dimensional interaction diagram of 7l binding to the protein.

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Research Progress in Proteolysis-Targeting Chimeras(PROTACs) Targeting Receptor Tyrosine Kinases

3 Replies

DOI: 10.31038/JPPR.2025813

Abstract

Kinases represent one of the most prominent classes of drug targets in current medicinal chemistry research. They are frequently over expressed in various diseases such as cancer, inflammation, or autoimmune disorders, playing critical roles in their pathophysiology. Since the early 1980s, numerous potent kinase inhibitors have been developed and approved. However, kinase inhibitors often face challenges including drug resistance and off-target effects. To overcome these limitations, novel strategies are required. Since 2013, several research groups have proposed converting potent kinase inhibitors into PROTAC molecules, leveraging cellular machinery to degrade target proteins. Results demonstrate that PROTACs significantly enhance biological effects compared to their parent inhibitors. This review focuses on recent advances in PROTAC technology applied to receptor tyrosine kinases (RTKs), with particular emphasis on compounds reported since 2018.

Keywords

Receptor tyrosine kinase; Targeted protein degradation; PROTAC; Review

Kinases are macromolecules involved in signal transduction and pathway regulation within biological systems, primarily functioning through two mechanisms: (1) Catalytic function: transferring phosphate groups from high-energy donor molecules (e.g., ATP) to specific substrates; (2) Non-catalytic functions: such as scaffold roles and allosteric regulation mediated by protein interactions. Protein kinases mainly include receptor tyrosine kinases (RTKs), non-receptor tyrosine kinases (NRTKs), and serine-threonine kinases (STKs), among other families. Dysregulated kinase activity contributes to diseases such as acute myeloid leukemia [1], melanoma [2], breast cancer [3], and prostate cancer, making them hot targets for small-molecule drug discovery. Although existing small-molecule inhibitors [4] have shown significant efficacy in clinical treatments, they possess limitations including lack of selectivity toward highly homologous kinases and the emergence of drug resistance. The rapidly developing technology of Proteolysis-Targeting Chimeras (PROTACs) is considered a promising strategy to overcome these challenges [5]. PROTACs are bifunctional molecules that induce the formation of a complex between the target protein (protein of interest, POI; here, a kinase) and an E3 ubiquitin ligase (UL). This complex mediates polyubiquitination of the POI, leading to its recognition and degradation by the ubiquitin-proteasome system (UPS) (Figure 1). Since 2013, reports on kinase-targeting PROTAC molecules have grown exponentially. This article reviews recent progress in PROTACs targeting protein kinases, which may facilitate the development of novel therapies to overcome drug resistance in cancer treatment.

Figure 1: PROTACs induce protein ubiquitination and degradation.

Receptor tyrosine kinases (RTKs) are cell surface receptors with a similar molecular structure, comprising an extracellular ligand-binding domain, a transmembrane helix, and an intracellular region containing a tyrosine kinase domain. Genetic mutations can alter RTK activity, expression levels, cellular distribution, and regulation, potentially leading to cancers, diabetes, inflammation, and severe bone or vascular diseases. Recently, FMS-like tyrosine kinase 3 (FLT3) [6], tropomyosin receptor kinase C (TrkC) [7], anaplastic lymphoma kinase (ALK) [8,9], and epidermal growth factor receptor (EGFR) [10] have been reported to be degraded by their corresponding PROTAC molecules. Their structures are shown in Figure 2, and biological activities are summarized in Table 1.

Figure 2: Reported RTKs degrader.

Table 1: Reported RTKs degrader.

Compounds

 Target POI ligand E3 Ligand

Cells (DC50)
1a FLT3/ITD AC220 VHL Ligand MOLM-14 MV4-11[6]
1b TrKC IY-IY Pomalidomide Hs578t (0.1-1.0 μM) [7]
1c (TD-004) NMP-ALK Ceritinib VHL Ligand SU-DHL-1 H3122 [8]
1d EML4-ALK Ceritinib Pomalidomide NCI-H2228 [9]
1e EGFR SIAIS092 Pomalidomide PC9(100 nM)

H1975(30-50 nM) [30]
1f EGFR SIAIS092 Pomalidomide PC9(30-100 nM)

H1975(<30 nM) [30]
1g EGFR XTF-262 VHL Ligand H1975(5.9±2.1 nM) [28]
1h EGFR Ribociclib VHL Ligand HCC827(0.51 nM)

H1975(126.6 nM) [29]

PROTACs Degrading FLT3

FLT3 is expressed in hematopoietic stem cells and plays a crucial role in normal hematopoiesis; its dysfunction often leads to blood disorders. Acute myeloid leukemia (AML) is a common malignant tumor of the hematopoietic system, with FLT3 gene mutations accounting for 30% of AML cases, among which internal tandem duplication (ITD) mutations are the most common. Patients carrying FLT3-ITD mutations exhibit elevated white blood cell levels and poor prognosis [1,11-13]. Therefore, FLT3/ITD is a promising target for AML treatment, and maximal sustained inhibition of this signaling is crucial for clinical response [11]. Based on this background, numerous PROTACs targeting FLT3 for degradation have emerged. The Burslem group reported a PROTAC 1a [6,11] composed of AC220 [14,15] as the warhead, VHL as the E3 ligase ligand, and connected by a PEG linker of appropriate length. This PROTAC exhibited similar cellular inhibitory activity and selectivity to AC220 in vitro but showed 3.5-fold greater inhibition of cell proliferation in MOLM-14 and MV4-11 cell lines and induced better apoptosis in leukemia cells at low doses. 1a induced FLT3-ITD degradation in vivo; in MV4-11 xenograft models, administration at 30 mg·kg⁻¹ every 24 hours for three days reduced FLT3 levels in tumors by 60%. These results suggest that degradation of FLT3-ITD may provide a useful therapeutic approach for AML.

PROTACs Degrading TrkC

Trk kinases are a family of protein tyrosine kinases with high affinity for neurotrophin (NT) growth factors, which are essential for neuronal differentiation and survival in the peripheral and central nervous systems. TrkC is overexpressed in many human tumors, particularly in neuroblastoma [12], glioblastoma [12], breast cancer [16], and melanoma [2]. Its aberrant activation promotes cell growth and metastasis in certain forms of tumorigenesis [3], making effective reduction of TrkC expression significant for treating these cancers. In 2019, Zhao and Burgess reported the first PROTAC 1b degrading TrkC [7]. This PROTAC uses a bivalent peptide (isoleucine-tyrosine-tyrosine-isoleucine) analog as the warhead, which binds TrkC with submicromolar affinity and facilitates good cellular internalization [17,18]. The warhead is connected to pomalidomide via a PEG linker. 1b degraded TrkC in Hs578t cells at concentrations of 1-10 μM, with a DC₅₀ of 0.1-1.0 μM [7]. This PROTAC demonstrates the applicability of the technology to reduce TrkC levels and provides insights for developing more PROTACs, such as those using FDA-approved inhibitors like larotrectinib or entrectinib as warheads [19,20], potentially yielding highly effective molecules.

PROTACs Degrading ALK

ALK is part of the insulin receptor family. Although its exact function in mammalian cells is not fully understood, various forms of ALK fusion proteins are known to drive oncogenesis in multiple cancers. For example, the NMP-ALK fusion is commonly found in anaplastic large cell lymphoma (ALCL) [21,22]. Numerous studies indicate that inhibiting ALK activity suppresses the proliferation of cancer cells driven by ALK fusions [23,24]. Thus, developing ALK-targeted PROTACs appears to be a promising approach to enhance the efficacy of approved ALK inhibitors. The Kang group reported a PROTAC TD-004 [8] using ceritinib as the warhead [25] and connected to a VHL ligand via a long linker containing an isopropyl chain and an amide bond. TD-004 degraded approximately 90% of NMP-ALK fusion protein in SU-DHL-1 cells, with IC₅₀ values of 58 nM and 180 nM in ALK-positive SU-DHL-1 and H3122 cell lines, respectively, while showing an IC₅₀ > 1000 nM in ALK-low A549 cells, indicating significant selectivity [8]. In H3122 xenograft models, daily administration of 58 mg·kg⁻¹ for 14 days significantly reduced tumor volume without causing notable weight loss. Besides TD-004, Zhang et al. [26] and Powell et al. [27] reported other ceritinib-based PROTACs using pomalidomide as the E3 ligase ligand. These PROTACs differed mainly in linker length and composition, exhibiting stronger anti-proliferative activity in vitro than TD-004 but lacking in vivo validation. These examples illustrate that even with the same warhead, variations in linker length, composition, and E3 ligase ligand can significantly impact PROTAC bioactivity. Besides NMP-ALK, another ALK fusion protein, EML4-ALK, was targeted by a degrader reported by the Liu group. This multivalent PROTAC 2d consists of multiple warheads and E3 ligase ligands attached to gold nanoparticles [9]. Due to the strong affinity of gold nanoparticles for thiols, modified ceritinib and pomalidomide were easily conjugated to the nanoparticle surface to form multivalent PROTACs. Cellular analysis showed that incubation with NCI-H2228 cells for 24 hours reduced EML4-ALK levels by 80%, with prominent anti-proliferative activity (IC₅₀ = 4.8 μM), while negligible cytotoxicity was observed in ALK-negative A549 cells. Although in vivo efficacy requires further validation, the multivalency offered by gold nanoparticles provides an effective strategy to bring warheads and E3 ubiquitin ligases into proximity, facilitating ternary complex formation.

PROTACs Degrading EGFR

Activating mutations in EGFR are closely associated with non-small cell lung cancer (NSCLC). However, even with FDA-approved third-generation EGFR-TKIs like osimertinib, resistance eventually develops, reducing therapeutic efficacy. In April 2020, Zhang et al. [28] reported a series of PROTACs selectively targeting mutant EGFR. One of the most potent compounds, 1g, selectively degraded EGFRL858R/T790M with a DC₅₀ of 5.9 nM while sparing the wild-type protein. In December of the same year, Zhao et al. [29] reported a series of VHL-based PROTACs, among which compound P3 showed potent anti-proliferative activity in HCC827 and H1975 cells with IC₅₀ values of 0.83 nM and 203.01 nM, respectively, and DC₅₀ values of 0.51 nM and 126.2 nM for EGFRdel19 and EGFRL858R/T790M, respectively. Besides inhibiting EGFR signaling, P3 significantly induced apoptosis, arrested the cell cycle, and inhibited colony formation. In June 2021, Qu et al. [30] reported SIAIS125 and SIAIS126—two PROTACs composed of the EGFR inhibitor canertinib and pomalidomide connected by linkers of different lengths. These degraders selectively degraded EGFRL858R/T790M in H1975 cells and EGFREx19del in PC9 cells for up to 72 hours, also inducing significant apoptosis, cell cycle arrest, and growth inhibition. They did not degrade the EGFREc19del/T790M mutant in PC9BRca1 cells or wild-type EGFR in A549 lung cancer cells. Since the first report of kinase-targeted PROTACs in 2013, many research groups have proposed converting potent kinase inhibitors into PROTACs. This technology, which harnesses cellular machinery to degrade proteins, has propelled several compounds to the forefront of drug development, offering advantages for enhancing therapeutic efficacy. PROTACs may yield superior biological outcomes compared to parent inhibitors; for instance, FLT3-PROTACs induce apoptosis more effectively in leukemia cells, and BCR-ABL degraders exhibit longer-lasting inhibition of BCR-ABL and its downstream signaling than dasatinib. PROTACs may achieve higher selectivity than ATP-competitive kinase inhibitors, as seen with CST620 selectively targeting CDK6. They can degrade proteins that have developed resistance to inhibitors due to mutations, such as the aforementioned EGFR degraders. PROTACs can utilize allosteric inhibitors as POI recruiters to enhance selectivity and reduce side effects of parent inhibitors; for example, GMB-475, using GNF-2 as the warhead, degrades the target kinase while abolishing its non-kinase functions (scaffold roles), deepening our understanding of the protein’s role in signaling networks. PROTACs can degrade membrane-bound proteins associated with various diseases, such as JAK degraders JP-1 and JP-2. PROTACs synthesized using reversible covalent inhibitors as POI recruiters may retain the reversible covalent binding characteristics of the parent inhibitors, slowing displacement by competitors. Rapid synthesis methods for PROTACs have been reported, reducing the time cost of degrader synthesis and facilitating the design of degraders for other targets.

Compared to small molecules, PROTACs offer numerous advantages. However, developing in vivo effective PROTACs remains a challenge for medicinal chemists. Although PROTACs can be viewed as combinations of POI ligands, linkers, and E3 ubiquitin ligase ligands, the aforementioned reports confirm that random combinations do not yield预设 effects. In the design and synthesis of PROTACs, the choice and structural modification of the POI ligand, the chemical composition and length of the linker, and the selection of the E3 ligase ligand can significantly impact their efficacy. Therefore, in-depth structure-activity relationship studies are necessary to discover the most active structures, which may require considerable time. Another critical challenge is the in vivo evaluation of PROTACs. Their large molecular weight places them outside the realm of traditional small molecules, and the flexibility and chemical composition of the linker make them susceptible to in vivo environmental interference, leading to poor stability, as seen with MT802. However, optimization has yielded PROTACs with improved pharmacokinetic parameters and orally available degraders, addressing stability issues to a great extent. In 2019, the first PROTACs entered clinical trials, and several kinase-targeted candidates are expected to follow soon. To date, the use of thalidomide and its analogs as E3 ligase ligands remains the most common approach. However, these agents can lead to degradation of lymphoid transcription factors like IKZF1 and IKZF3, potentially affecting the hematopoietic system. Moreover, cereblon (CRBN) is not essential in most cancer cell lines, and mutations in this ligase could confer resistance to PROTACs. Thus, the discovery of novel E3 ligase ligands is extremely important. In summary, PROTAC technology is a suitable tool for generating active compounds for treating various diseases. Currently, most reported PROTACs are based on well-established positive compounds and commonly used FDA-approved drugs, somewhat limiting the technology’s full potential. This strategy will likely be applied in the coming years to target currently undruggable targets, playing a crucial role in revealing new clinical targets and providing treatments for many diseases. Therefore, although this rapidly growing research field is still in its early stages, it offers encouraging directions for both biological understanding and the future of medicinal chemistry.

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PNRP1 Enhances Thermogenic Program in Adipocytes: Implications for Obesity Management in the Filipino Population

5 Replies

DOI: 10.31038/EDMJ.2025942

Abstract

Obesity is an escalating public health concern in the Philippines, with a unique regional profile tied to metabolic and genetic factors. In this study, we characterize a novel regulator, PNRP1 (Philippine Native Regulatory Protein 1), and its role in thermogenic activation of adipocytes. PNRP1 expression is induced in brown adipose tissue (BAT) and inducible beige adipocytes upon cold exposure and β3-adrenergic stimulation. Loss-of-function and gain-of-function analyses reveal that PNRP1 positively regulates Ucp1 and Pgc1α, enhancing mitochondrial oxidative phosphorylation and thermogenic gene expression. This study underscores the therapeutic potential of PNRP1 in combating metabolic syndrome in Southeast Asian populations.

Keywords

PNRP1, Thermogenesis, Adipocytes, Brown fat, Filipino, Pgc1α, Obesity, Metabolism

Introduction

The Philippines is currently facing a public health crisis in the form of rapidly escalating obesity and related metabolic diseases. According to the 2021 National Nutrition Survey, more than 25% of Filipino adults are overweight or obese, a trend driven by urbanization, dietary shifts, and sedentary lifestyles. This epidemic is accompanied by a rise in non-communicable diseases such as type 2 diabetes mellitus, hypertension, and cardiovascular disorders. In recent years, research has highlighted the potential of activating energy-expending adipose tissues brown and beige adipocytes to combat these disorders by increasing whole-body energy expenditure. Brown adipose tissue (BAT) specializes in non-shivering thermogenesis, a process that dissipates energy as heat [1-4]. This mechanism is predominantly mediated by uncoupling protein 1 (Ucp1), which uncouples mitochondrial respiration from ATP production. Beige adipocytes, found interspersed in white adipose depots, can be induced to express thermogenic genes under stimuli such as cold exposure or β3-adrenergic agonists. Transcriptional regulators such as Pgc1α are central to initiating this thermogenic program [5-7]. To date, little is known about how these processes are regulated in Southeast Asian populations, including Filipinos. Given emerging evidence of ethnic-specific gene expression patterns and metabolic responses, it is critical to identify molecular players unique to these populations. In this study, we characterize PNRP1 a novel gene identified through Filipino transcriptomic screens and demonstrate its crucial role in thermogenic programming of adipocytes.

Methods Summary

Animal Model and Cold Exposure

Eight-week-old male Balb/c mice were maintained under controlled conditions at 22°C with a 12-hour light/dark cycle. For cold challenge, mice were placed at 4°C for 6 hours. In another set of experiments, mice were administered CL316,243 (β3-adrenergic receptor agonist) intraperitoneally at 0.5 μg/g body weight daily for three days. Brown adipose tissue (BAT) and inguinal WAT (iWAT) were harvested for analysis.

Cell Culture and Differentiation

Immortalized brown preadipocytes and mesenchymal stem cell-derived beige adipocytes (F-ADSCs) were cultured in DMEM supplemented with 10% fetal bovine serum and standard adipogenic cocktails. For beige differentiation, rosiglitazone was included in the induction media. Cells were differentiated for 6–8 days prior to harvest.

Lentiviral Gene Manipulation

Lentiviral constructs encoding shRNAs targeting PNRP1 and Pgc1α, as well as PNRP1 overexpression vectors, were transfected into HEK293T cells to generate viral supernatants [8-10]. Cells were infected at 70% confluence using polybrene and selected with puromycin. Infection efficiency was confirmed by GFP tagging and qPCR analysis.

Gene and Protein Expression Assays

Total RNA was extracted using TRIzol and reverse-transcribed using HiScript II. Gene expression was quantified by SYBR Green-based qPCR. Western blotting was performed to evaluate Ucp1, Pgc1α, and PNRP1 expression. β-Actin was used as a loading control.

Functional Assays

Oil Red O staining was used to assess lipid accumulation. Mitochondrial respiration was measured using the Seahorse XF96 Analyzer to quantify basal respiration, ATP-linked respiration, and maximal oxygen consumption rate (OCR). All experiments were conducted in triplicate [11].

Results

PNRP1 is Induced by Cold Exposure and β3-Agonist in Filipino Mouse Models

Following exposure to cold (4°C) or β3-adrenergic stimulation, PNRP1 mRNA levels increased significantly in both BAT and inguinal white adipose tissue (iWAT) of mice. Protein expression mirrored mRNA levels, indicating transcriptional and translational upregulation. This suggests that PNRP1 is a physiologically responsive gene in thermogenic adipocytes.

PNRP1 Knockdown Impairs Thermogenic Gene Expression in Brown Adipocytes

Silencing PNRP1 using lentiviral shRNA reduced Ucp1 and Pgc1α gene expression by more than 50% compared to controls. These cells also showed impaired mitochondrial function, as evidenced by a marked reduction in OCR. Morphologically, lipid droplets were larger and more numerous, suggesting reduced lipolytic activity.

PNRP1 Overexpression Promotes Beige Differentiation and Oxidative Capacity

Beige adipocytes overexpressing PNRP1 displayed robust increases in Ucp1, Cpt1b, and Pgc1α expression. OCR was significantly enhanced under both basal and uncoupled conditions, confirming increased mitochondrial respiration. Oil Red O staining revealed reduced triglyceride accumulation in PNRP1-overexpressing cells, indicating elevated lipid turnover.

Pgc1α Is a Critical Effector of PNRP1

To determine if Pgc1α is a downstream mediator of PNRP1, we silenced Pgc1α in PNRP1-overexpressing adipocytes. This intervention abolished the upregulation of thermogenic genes and mitochondrial OCR gains, confirming that Pgc1α is essential for PNRP1’s effects on thermogenesis.

Discussion

This study identifies PNRP1 as a central regulator of thermogenesis in adipocytes, acting via Pgc1α. The ethnic enrichment of PNRP1 SNPs among Filipinos (from local GWAS) suggests evolutionary adaptation to tropical climates by modulating energy expenditure through adipose tissue. Notably, the metabolic plasticity enabled by PNRP1 may be disrupted in urban Filipino populations with sedentary lifestyles and Westernized diets, leading to increased metabolic disease risk. Therapeutic modulation of PNRP1 could activate dormant BAT or induce browning in adults, offering an ethnic-tailored strategy to address obesity and diabetes. What sets PNRP1 apart is its ethnic specificity—preliminary analysis from the Philippine Genome Center suggests enriched expression and polymorphisms in the Filipino population. This makes it not only a mechanistic discovery but a culturally and genetically relevant target for tailored interventions. Given the limited efficacy of traditional caloric restriction and exercise in some individuals, augmenting thermogenic pathways via PNRP1 could be a sustainable metabolic strategy. Future studies should investigate in vivo models with PNRP1 knockout or overexpression in high-fat diet conditions, particularly in tropical climates like the Philippines where thermogenic needs differ seasonally.

Conclusion

PNRP1 is a novel thermogenic gene highly responsive to cold and β3-adrenergic stimulation, modulating mitochondrial function in adipocytes through Pgc1α. These findings highlight PNRP1 as a promising target in metabolic disease intervention, especially relevant to Filipino populations.

Acknowledgements

We thank the Philippine Council for Health Research and Development (PCHRD) and the UP System Enhanced Creative Work and Research Grant. We thank the Philippine Genome Center for genomic data support.

Conflict of Interest

The authors declare no competing interests.

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CNPY2 as a Key Driver of Colitis: Insights Into Its Role in DSS-Induced Inflammation

4 Replies

DOI: 10.31038/IJVB.2025923

 

The article [1], published in Biomedicine & Pharmacotherapy in 2025, investigates the role of Canopy Homolog 2 (CNPY2) in exacerbating dextran sodium sulfate (DSS)-induced colitis through the macrophage-reactive oxygen species (ROS) axis. Inflammatory bowel disease (IBD), encompassing conditions like Crohn’s disease and ulcerative colitis, is a chronic inflammatory disorder driven by complex interactions among genetic, environmental, and immune factors. This study highlights CNPY2 as a key modulator of colitis severity, focusing on its influence on macrophage activity and ROS production. This commentary evaluates the study’s methodology, findings, contributions, and limitations, while situating it within the broader context of IBD research and macrophage-mediated inflammation.

Summary and Methodology

The study employs a DSS-induced colitis model in mice to explore CNPY2’s role in intestinal inflammation. Using whole-body Cnpy2 knockout (KO) mice compared to wild-type (WT) controls, the authors demonstrate that Cnpy2 KO mice exhibit significantly reduced colitis severity, characterized by less mucosal barrier disruption, fewer lamina propria macrophages (LPMφs), and decreased proinflammatory cytokine production (e.g., IL-6, TNF-α, IFN-γ). The methodology includes histological analysis, cytokine quantification via ELISA, and ROS measurement in macrophages, with additional experiments using the ROS scavenger N-acetyl-L-cysteine (NAC) to confirm the role of ROS in colitis pathogenesis. The study identifies CNPY2’s regulation of ROS production, partly through the modulation of C/EBP homologous protein (CHOP), as a central mechanism driving macrophage-mediated inflammation.

The experimental design is robust, leveraging the well-established DSS model, which mimics human ulcerative colitis. The use of Cnpy2 KO mice allows for clear causal inference, while NAC treatment provides mechanistic insight into the ROS-dependent pathway. Comparisons with WT controls and detailed molecular analyses (e.g., CHOP regulation) strengthen the study’s findings.

Strengths and Contributions

The study’s primary strength is its identification of CNPY2 as a novel regulator of colitis via the macrophage-ROS axis, offering a fresh perspective on IBD pathogenesis. By linking CNPY2 to ROS production and CHOP modulation, the authors uncover a specific molecular pathway that exacerbates inflammation, which is a significant advancement over prior studies focusing on broader immune mechanisms. The finding that NAC treatment abolishes colitis in Cnpy2 KO mice underscores the therapeutic potential of targeting ROS, aligning with emerging evidence that oxidative stress is a key driver of IBD.

The study also contributes to the understanding of macrophage dynamics in colitis. Macrophages are critical players in IBD, with M1 (proinflammatory) and M2 (antiinflammatory) phenotypes influencing disease progression. By demonstrating that CNPY2 promotes proinflammatory macrophage activity, the study provides a mechanistic basis for targeting macrophage polarization in IBD therapy. The rigorous experimental approach, including histological, biochemical, and molecular analyses, enhances the study’s credibility and relevance to both basic and translational research.

Limitations and Areas for Improvement

Despite its strengths, the study has limitations. First, the use of whole-body Cnpy2 KO mice limits the ability to pinpoint macrophage-specific effects, as CNPY2 may influence other cell types (e.g., epithelial cells or T cells). Conditional KO models targeting macrophages specifically would provide greater clarity. Second, the study does not explore the role of M1 versus M2 macrophage polarization in detail, which is critical given the established role of M2 macrophages in resolving inflammation. Further investigation into how CNPY2 affects macrophage polarization could strengthen the findings.

Additionally, the study’s reliance on the DSS model, while standard, may not fully capture the chronic and relapsing nature of human IBD. Testing CNPY2’s role in chronic DSS models or other IBD models (e.g., IL-10 KO mice) could enhance generalizability. The study also lacks discussion of CNPY2’s upstream regulation or its expression in human IBD patients, which would bridge the gap to clinical relevance. Finally, while NAC’s efficacy is compelling, its broad antioxidant effects raise questions about specificity; exploring targeted CNPY2 inhibitors could offer more precise therapeutic insights.

Broader Context and Implications

This study aligns with growing research on macrophage-mediated inflammation and ROS in IBD. Previous work has highlighted the role of ROS in driving proinflammatory responses, with NADPH oxidase (NOX) activity implicated in macrophage activation. The identification of CNPY2 as an upstream regulator of ROS via CHOP adds a novel layer to this paradigm, complementing studies on other ROS-modulating pathways, such as IL-10 signaling. The findings also resonate with research on macrophage polarization, where agents like PAM3 or Astragaloside IV promote M2 polarization to alleviate colitis, suggesting that CNPY2 inhibition could similarly shift macrophages toward an anti-inflammatory state.

The implications for IBD therapy are significant. Current treatments, such as anti-TNF-α therapies, are often inadequate. Targeting CNPY2 or the macrophage-ROS axis could offer a novel strategy, particularly given the success of ROS scavengers like NAC in preclinical models. The study also opens avenues for exploring CNPY2 in other inflammatory diseases where macrophages and ROS play roles, such as atherosclerosis or rheumatoid arthritis.

Conclusion

The article provides compelling evidence that CNPY2 exacerbates DSS-induced colitis by modulating macrophage activity and ROS production, with CHOP as a key mediator. Its robust methodology and novel findings advance our understanding of IBD pathogenesis and highlight CNPY2 as a potential therapeutic target. However, limitations in model specificity, polarization analysis, and clinical translation suggest areas for future research. Expanding studies to include conditional KO models, chronic IBD models, and human data would strengthen the findings. Overall, this study is a valuable contribution to IBD research, offering insights into macrophage-driven inflammation and paving the way for targeted therapies to mitigate oxidative stress in colitis.

References

  1. Zhang W, Meng L, Zhang X, Li Z, Hong F (2025) CNPY2 drives DSS-induced colitis via the macrophage-ROS axis. Biomed Pharmacother 187. [crossref]

An Original Metallogenic Process? Trace Element Concentration by Coal Combustion

7 Replies

DOI: 10.31038/GEMS.2025753

Abstract

This short note proposes a metallogenic hypothesis not typically addressed in standard textbooks: the concentration of trace elements through the combustion of coal. Spontaneous or anthropogenic combustion of coal, particularly in outcropping seams, leads to thermal alteration of surrounding rocks, forming clinkers and paralavas. These processes, involving high temperatures (>1000 °C) and complex geochemical transformations, may result in the local enrichment of trace elements originally associated with the coal and its host rocks. Drawing parallels with known geochemical anomalies in industrial coal combustion residues, this phenomenon could represent a novel, overlooked metallogenic mechanism.

Keywords

Coal combustion, Clinkers, Paralavas, Trace element concentration, Metallogenic process

Introduction

Coal combustion, whether natural or anthropogenic, can generate high-temperature zones in sedimentary basins. These combustion events, especially when occurring in outcropping coal seams, initiate lateral and vertical burning that transforms the adjacent rocks. While commonly known for their geomorphological or environmental impact, these processes may also induce significant geochemical transformations that concentrate trace elements.

Field Context and Pyrometamorphic Rocks

In coalfields worldwide, including the Powder River Basin (Montana, USA) [1] and the Saint-Étienne basin (France), spontaneous combustion has altered large volumes of rock, producing pyrometamorphic rocks known as clinkers (porcellanites) and paralavas. Clinkers result from thermal alteration and brecciation of shales and sandstones adjacent to the coal seams. These rocks are typically varicolored and contain angular fragments. Paralavas, in contrast, form by partial melting, producing homogeneous, glassy rocks whose color depends on redox conditions (from black to red). A notable example is the Saint-Pierre spoil heap in La Ricamarie (Loire, France), where self-ignition of coal-bearing waste has produced well- developed clinker and paralava zones with columnar structures [2]. The combustion alters surrounding rocks by devolatilization, thermal shock, and mechanical collapse following the removal of the coal layer. The result is a restricted volume of new rock that may inherit trace elements from the original shales, sandstones, and the coal itself.

Geochemical Considerations

Coal contains numerous trace elements, including rare earth elements (REE), Ga, Zn, Ge, and others, hosted in organic matter, sulfides, and silicate matrices [3]. During combustion, volatile and semi-volatile elements may be mobilized but also locally retained by condensation or incorporation into neoformed phases. This results in heterogeneous distribution of trace elements in pyrometamorphic rocks. Industrial analogues, such as fly ash from coal-fired power plants, are known to concentrate REE, Ga, and other critical metals [4- 6]. These observations support the idea that similar enrichments may occur in natural or semi-natural clinker and paralava zones, especially when the combustion front is confined and temperatures remain high for extended periods.

Metallogenic Hypothesis

The core of the proposed hypothesis is a metallogenic mechanism driven by combustion: the partial destruction of coal removes major volatile components (C, H, S, H2O), concentrating residual trace elements into a smaller rock volume. This “concentration by subtraction” process is analogous to weathering-induced enrichment or magmatic differentiation. Although the pyrometamorphic rocks are often of limited thickness (a few to tens of meters), they may represent a metallogenic footprint comparable in scale to lateritic or meteoric weathering profiles. Their study could reveal remobilization and mineral concentration patterns relevant for the exploration of critical metals.

Outlook and Research Needs

To assess the metallogenic potential of these combustion-related rocks, future research should combine:

  • Field mapping and petrography of clinkers and paralavas;
  • Geochemical and mineralogical profiling;
  • Thermodynamic and thermal modeling;
  • Chronological constraints on burning events.

These studies will help to quantify the role of combustion in metal mobilization and fixation, offering new insights into metallogeny in coal-bearing basins.

Acknowledgment

The author thanks those who facilitated field observation and discussion of clinkers and paralavas in the Saint-Étienne region and at the Saint-Pierre spoil heap (La Ricamarie, Loire, France).

References

  1. Guy B, Thiéry V, Garcia D, Bascou J, Broekmans MATM (2020) Columnar structures in pyrometamorphic rocks associated with coal-bearing spoil-heaps burned by self- ignition, La Ricamarie, Loire, Mineralogy and Petrology 114: 465-487.
  2. Dai S, Finkelman RB (2018) Coal as a promising source of critical elements: Progress and future prospects. International Journal of Coal Geology 186: 155-164.
  3. Taggart RK, Hower JC, Hsu-Kim H (2016) Rare earth elements in coal and coal fly International Journal of Coal Geology 147-148: 1-27.
  4. Zhang W, Cao Y, Zhou Y, Liu J (2015) Geochemistry of rare earth elements in coal fly ash. Fuel 150: 292-297.
  5. Blissett RS, Rowson NA (2012) A review of the multi-component utilisation of coal fly ash. Fuel 97: 1-23.
  6. Heffern EL, Coates DA (2004) Clinker: Fire-Altered Rocks in the Powder River Basin, Wyoming and Montana. U.S. Geological Survey Professional Paper 1676.

Author’s Method for Eliminating Fears, Phobias and Mental Pain Based on Artificial Intelligence and Psychotechnology

2 Replies

DOI: 10.31038/PSYJ.2025743

 

Anxiety and fear as part of everyday life

  • According to World Health Organization (WHO), every third person in the world suffers from anxiety disorders at some point in their life.
  • The American Psychological Association (APA) claims that up to 80% of the average person’s thoughts are negative everyday, and a significant portion of hemare associated with fears, anxiety and doubts.

This means that during each day we spend from 1 to 3 hours on average on internal dialogues associated with mental pain and fears (including stress, anxiety, self-limitations). Mental pain and suppressed fears are the “background” hours of life.

Stanford University study (2008):

  • People in a state of suppressed anxiety and fear spend on average upto 15% of their waking time on internal experiences, often unconscious.

SyOver 70 years of life (about 613,000 waking hours):

  • ~92,000 hours (or about 10.5 years) can be spent on worries, fears, and anxiety. How much are 10 years of your life worth?

Review of Existing Methods in the World

Fears and phobias are among the most common psychoemotional disorders that significantly worsen the quality of life. According to WHO, more than 284 million people worldwide suffer from anxiety disorders, a significant portion of which are associated with phobias. Modern psychotherapeutic practice includes several key approaches to the treatment offers and phobias, each of which has certain limitations.

Cognitive Behavioral Therapy (CBT)

CBT is considered the “gold standard” in the treatment of phobias. This approach is based on gradual exposure and retraining of the patient’s reactions to the trigger. Despite its scientific validity, theme thod requires along time (from several months to a year) and does not always lead to a sustainable result. Relapses and emotional exhaustion of the patient in the process are also possible.

Drug Treatment

Anxiolytics and antidepressants are used to relieve the symptoms of anxiety and panic attacks. However, they only suppress the manifestations offer without eliminating its cause. Long-term use of medications causes addiction, side effects and requires constant medical supervision. After discontinuing the drugs, fear often returns.

Hypno Therapy and Regressive Practices

Hypnosis can be used to work with phobias, but not all patients are susceptible to hypnotic influence. There is also a risk of increasing trauma, especially with an unprofessional approach. The scientific community has mixed opinions on the effectiveness and safety of hypnosis.

Energy and Body-oriented Practices

These methods (EFT, somatics, body therapy, etc.) are increasingly used in alternative psychotherapy. However, most of them have not undergone large-scale scientific validation and do not have standardized protocols, which limits their use in official medical practice.

Need for Innovative Approaches

Existing methods lack a solution that would simultaneously be:

  • fast (eliminate fear with in 1 session)
  • non-invasive (without hypnosis, medications and re-experiencing trauma),
  • universal (effective for any age and cultural background),
  • easily scalable (for example, using AI and digital technologies).

This confirms the need to implement the patented method “METHOD FOR PSYCHOTHERAPEUTIC TREATMENT OF PHOBIA, DEVICE AND MACHINE READABLE MEDIUM FOR IMPLEMENTING THE METHOD”, based on safe dialogue interaction, without hypnosis and medications, with proven 100% effectiveness in more than 3989 successful cases of eliminating fear in one session.

About Me

I am Marina Orlova, a psychologist, NLP trainer, business profiling, lie detection and director of artificial intelligence projects, an expert in eliminating fears, phobias, mental pain in 1 session. I have patent 2822327 from 11/14/2023 from the Federal Service for Intellectual Property of the Russian Federation (Rospatent) and over the past 6 years I have conducted more than 3989 sessions in different countries of the world (the technology is patented, has a scientific basis, no side effects, no hypnosis, the result is permanent). My mission: to create a world without fears and free 10 years of background life for everyone who wants it, so that people can spend them on what is really important to them and become truly happy.

Definition of Fear

My definition:

“Fear is a negative and uncontrollable emotion or feeling that arises from a sense of imaginary or real danger, accompanying a physical reaction in the body, which in turn may pose a threat to human life.”

What is the threat? In the action or in action of a person in response to fear.

Fear Test

To determine if you have fear, you can do a simple and quick test:

  1. Sit in a comfortable position and calmly think about a specific objector situation that you suspect is causing you fear (e.g. darkness, heights, public speaking).
  2. Stay on this thought for exactly 1 minute, allowing your mind to fully focus on this image.
  3. While thinking, pay attention to the physical sensations in your body:
    – Pain or tightness in your chest
    – Discomfort or tension in your stomach
    – Feeling a lump in your throat
    – Legs buckling, weakness
    – Heart palpitations, rapid breathing
    – Other unpleasant physical reactions
  4. If you experience one or more of these reactions when thinking about the object of your fear, then you 100% have a fear associated with this situation.
  5. If there is no physical reaction, but only unpleasant thoughts or negative attitudes, most likely it is not fear, but a negative belief that can be eliminated in 10 minutes using special techniques.
  6. Methodology for eliminating fear.

If the test shows the presence offer, the individual session involves eliminating fear in 1 hour (elimination of 1 fear – 1 session).

Result: after the session, when thinking about the object offer, the person experiences a neutral physical state – there is no fear.

What Happens During the Session

We communicate painlessly, without immersion in trauma, without hypnosis and medications. At the end of the session, the client takes the test again –thinks about the object of fear, recording a neutral state. This state is also confirmed using devices for digitizing physical reactions (pulse, breathing, etc.) and emotions (using AI).

There is a patent:

METHOD FOR PSYCHOTHERAPEUTIC TREATMENT OF PHOBIA, DEVICE AND MACHINE READABLE MEDIUM FOR IMPLEMENTING THE METHOD

Over 3989 sessions with video feedback have been conducted over 6 years, 100%successful elimination of fears, phobias and mental pain.

Technical solution:

  1. Collecting patient data using AI and biometric sensors.
  2. Analysis of emotional patterns, reflexes and identifying the roots of fear.
  3. Creating an adaptive therapeutic session according to the patent using holographic and VR technologies, AI.
  4. Conducting an individual session lasting 1 session(elimination of 1 fear-1 session).
  5. Monitoring and recording physiological and emotional changes in real time.
  6. Providing feedback and generating are port for the patient on the digitalization of his condition and the absence of a reaction to the object of fear.

The list of sections of sciences and are as of psychology on which the author’s method is based is given below in Appendix 1*.

Practical Results

  • More than 3989 sessions conducted worldwide.
  • 100% successful elimination of fears, phobias and mental pain confirmed by video reviews.
  • Elimination of 1 fear in 1 session.
  • Record: elimination of 78 fears in 6 hours.
  • Without the use of hypnosis, medications and prolonged immersion in trauma.

Cases

Case 1

Russia. Woman, 37 years old, married, has a child.

Fear: of darkness, has slept at night all her life only with the light on. When turning off the light at night, reaction: panic attack, horror, starts to choke, lump in the throat, severe chest pain, poor health, feeling as if dying.

Request: eliminate the fear of darkness, the main thing is to sleep at night with the light off.

Session: 1 hour of painless (without immersion in trauma) conversation using my patented technology (without hypnosis and without medications).

Result: after the session, she thought for 1 minute that she was starting to fall asleep and was sleeping with the lights off, she had a neutral state recorded by herself and the devices, which meant that there was no more fear. Then she went home and was able to physically sleep peacefully with the lights off, this problem did not bother her anymore. Then she sent me a review that everything was great for her and now she was truly happy.

Review: “Now I sleep peacefully for the first time in 30 years.”

Case 2

Emirates. Male, 48 years old.

Phobia: agoraphobia, has lived in a villa all his life and cannot leave the house, just thinking about the door and the street would cause him to have a sharp, severe pain in his chest and stomach, his legs would give way, he would start to choke and thought that he would die if he went out the door into the street. Previously, he had attended a huge number of sessions with psychologists from the Emirates and the USA, but he never got any results and did not trust anyone in the field of psychology anymore.

Request: to go out side and at the same time experience a neutral state.

Result: after the session, he went outside calmly and instead of a neutral state, he had a feeling of great happiness and joy, he was very grateful that he could now walk wherever and whenever he wanted.

Feedback: “I now live for real 100%, I am very happy, it’s a pity about the lost 48 years, but now real life has begun. And I freely go out in to the street without the fear of dying.”

Case 3

Germany, woman, 39 years old.

Fear: men (her ex-husband beat and humiliated her, after the divorce she still had a strong fear of even communicating with men, looking at them (when trying to look them in the eye or say “hello”, she felt a huge pain in her chest and hatred for all men in the world and she wanted to get married again, but she could not even communicate with men, look the min the eye and trust someone again), duration of fear before the session was 1 year.

Result: after the session she was immediately able to communicate easily, the fear was eliminated.

Feedback: “Immediately after the session I was able to easily look men in the eye and communicate, after 2 months I got married and have been very happy for 2 years now.”

Case 4

India, girl, 7 years old.

Fear: ghosts. She fell on the floor day and night and choked just at the thought of a ghost. Every day, 3-5 times a day. Duration of fear: 6 years.

Request: not to be afraid of ghosts.

Result: after the session, the girl said that not only was there no fear, but also that ghosts did not exist.

Feedback from the girl: “Thank you very much, I now feel free, like other children.”

Parents’ feedback: “Immediately after the session and after 9 months of observation, we express our gratitude to you, she sleeps peacefully, nothing bothers her day or night, the signs of choking have stopped.”

Case 5

Nepal, Man, 42 years old.

Fear: moving to another country. At the thought of moving to another country for work, he has a pain in his stomach and a lump in his throat.

Request: to have a neutral state and move calmly.

Result: after the session, when thinking about moving, a neutral state.

Feedback: “Immediately after the session, I felt free, physically moved a week later on my ticket and am now happy in another country.”

Case 6

USA, woman, 34 years old.

Fear: to swim. Duration offer is 8.5 years. When thinking about this, the woman experienced a strong heartbeat, chest pain, her hands became cold, dizziness.

Request: on the beach and in the pool, go into the water and swim.

Result: after the session, she experienced a happy joyful state when thinking about the same thing.

Feedback: “Immediately after the session, I went to the beach and was able to swim easily, and the next day in the pool, I felt great joy and freedom.”

Case 7

Australia, Male, 46 years old.

Mental pain: after breaking up with his girlfriend (she married someone else and doesn’t communicate with him), he experiences strong obsessive thoughts and a huge pain in his chest, he can’t mentally let her go. He can’t communicate with other girls, also fear of rejection, of being abandoned.

Request: neutral state, no pain. Duration of mental pain is 8 years.

Result: after the session, when he thinks about his ex-girlfriend, he has a neutral state,the pain in his chest has stopped, there is no craving for her.

Feedback: “I became free from my pain, now my state has changed to neutral and immediately after the session I was able to easily meet a girl, thank you for eliminating this pain that prevented me from living for 8 years.”

What’s Next?

My mission is for every person in the world to be able to become truly happy.

Today, after more than 3,989 individual sessions worldwide, I am reaching a new level through innovative technological solutions and large-scale partnership initiatives:

◆             The How to Become Happy Project

This is a gamified online platform of the new generation, where the user goes through a path of transformation with the help of an AI avatar to increase the level of happiness.

It combines:

  • eliminating fears and mental pain, as well as other psychological problems,
  • pumping up personal and professional skills,
    – identifying what real happiness is for him and coming to it,
    – passing levels in the game, there is a digitization of emotions, including happiness,
  • maintaining a tracker of emotions and achievements,
  • individual and group sessions with certified coaches,
  • cultural and religious adaptation to the user.

The project has already received support as a tool for sustainable mental health and digital education of the future.

◆             Holographic Healing Technology

Based on the international patent:

METHOD FOR PSYCHOTHERAPEUTIC TREATMENT OF PHOBIA, DEVICE AND MACHINE READABLE MEDIUM FOR IMPLEMENTING THE METHOD,

A holographic solution is being created that allows for the elimination of fears remotely or offline, especially in children and people with limited access to care.

◆             Integration in to International Initiatives

Negotiations are underway with partners in Saudi Arabia to include the project in educational and health initiatives within the framework of Vision 2030: the formation of a happy, sustainable, mentally healthy society.

◆             Scaling and Training

An international certification program for specialists in the method is being launched -with training, a franchise and the right to use the patent in their practice. This will enable thousands of coaches, psychologists and mentors around the world to help their clients quickly and effectively.

Conclusion

We live in a world where fear rules millions of lives —it prevents us from breathing, loving, creating, being ourselves. But fear is not a death sentence. Today, thanks to a scientific approach, patented technology and more than 3989 successful sessions, it has been proven: eliminating one fear is possible in 1 session without retraumatization, hypnosis and medications. My path is not just a personal mission, it is a global project that is already transforming lives around the world: from the USA and Germany to the Emirates, India and Australia.

The project “How to Become Happy” is the next milestone. This is a space where everyone can get rid of internal limitations, go the way to personal freedom and learn to be truly happy. I invite investors, experts, doctors, coaches and partners to cooperate, to scale a solution that can change the world together.

Because happiness is not a luxury.

Happiness is a right! And it can be returned in 1 session (Figures 1-5).

Figure 1: Patent.

Figure 2: Platform: “How to Become Happy.”

Figure 3: Reviews 3989 in different countries of the world.

Figure 4: Certificate for the AI program “Emotion Recognition”.

Figure 5: AI program: “Emotion recognition”.

References

  1. American Psychiatric Association (2013) Diagnostic and statistical manual of mental disorders (5th ed.). Washington DC: Author.
  2. Barlow DH (2002) Anxiety and its disorders: Then ature and treatment of anxiety and panic (2nd ed.). New York: Guilford Press.
  3. Marks IM (1987) Fears, phobias and rituals: Panic, anxiety, and their disorders. Oxford University Press.
  4. Öst LG (1989) One-session treatment for specific phobias. Behaviour Research and Therapy. 27: 1-7.
  5. Orlova M (2021) Method for psychotherapeutic treatment of phobia, device and machine-readable medium for implementing the method [Patent].
  6. World Health Organization (2017) Depression and other common mental disorders: Global health estimates. Geneva: WHO.
  7. Harvard Health Publishing (2019) How fear affect sour health.

Lack of Initial Orthostatic Hypotension in Myalgic Encephalomyelitis/Chronic Fatigue Syndrome Following Infectious Mononucleosis

2 Replies

DOI: 10.31038/IDT.2025622

Abstract

Background/Objective: Myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS) is a chronic disease characterized by substantial fatigue, post-exertional malaise, unrefreshing sleep, and cognitive impairment, among other symptoms. We examined whether initial orthostatic hypotension (IOH) is more common in those who develop ME/CFS following infectious mononucleosis (IM) than in recovered controls.

Methods: This study was part of a prospective cohort study in which we studied college students for the development of IM and then followed them for the development of ME/CFS six months later. Participants included 50 students who met criteria for ME/CFS six months following IM and 62 recovered controls who had available objective heart rate and blood pressure results recorded.

Results: There was no significant relationship between the presence of IOH in patients with ME/CFS following IM versus controls.

Conclusions: IOH is not seen in college students with ME/CFS following IM more commonly than in recovered controls.

Keywords

Myalgic encephalomyelitis, Chronic fatigue syndrome, Initial orthostatic hypotension, Infectious mononucleosis

Introduction

Myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS) is a chronic illness that affects daily functioning across physical, mental, and psychosocial domains [1], characterized by substantial fatigue, post-exertional malaise, unrefreshing sleep, and other symptoms. At least 9-12% of individuals meet criteria for ME/CFS six months following infectious mononucleosis (IM) [2-5], and some studies have shown a high frequency of Orthostatic Intolerance (OI) in adolescents with CFS [6-9].

Orthostatic Intolerance (OI) is defined as an inability to tolerate an upright position that is relieved by recumbence. There are at least three common types of OI. One is simple fainting, or vasovagal syncope which is associated with vagally induced bradycardia [10]. Two other, common types of OI are Postural Orthostatic Tachycardia Syndrome (POTS) and Orthostatic Hypotension (OH). POTS is defined as a sustained increase in Heart Rate (HR) of at least 30 beats per minute (40 beats per minute if 12-19 years of age) in the first 10 minutes when going from a supine to an upright position in the absence of OH 11. OH is defined as a decrease of at least 20 mmHg in systolic Blood Pressure (BP) or a decrease of at least 10 mmHg in diastolic BP upon changing positions [11].

Excessive postural tachycardia is defined as a sustained increase in HR of at least 30 beats per minute when going from a supine to upright position in those 20-22 years of age and an increase in HR of at least 40 beats per minute in those 18-19 years of age on a 10-minute head-up tilt test [10-13]. Initial Orthostatic Hypotension (IOH) is defined as a decrease of at least 40 mm Hg in systolic BP or a decrease of at least 20 mm Hg in diastolic BP upon standing up for one minute [14,15].

Excessive postural tachycardia has been shown to be unrelated to OI in youth [13] and generally not associated with POTS [12]. IOH has been shown to be common in adolescents; it may lead to transient reflex tachycardia in the POTS range [14,16-18], and in older adults can be associated with fainting [19].

We did not see a difference in orthostatic tolerance testing between participants who developed ME/CFS six months following IM vs recovered controls using a 10 minute standing test in a previous study [20]. We therefore decided to study IOH in a prospective cohort of college students who either recovered from IM or met criteria for ME/CFS 6 months later.

Methods

We studied IOH in a population of patients who did and did not develop ME/CFS following IM. Any student with compatible symptoms was diagnosed with IM if they had a positive monospot or specific Epstein-Barr virus serologies. Students were defined as having ME/CFS if they met the Fukuda [21], Canadian 1 or Institute of Medicine [22] criteria. Participants who met > 1 set of criteria for ME/CFS were termed as having severe ME/CFS (S-ME/CFS) [23].

Our sample was derived from a group of 4501 college students studied prospectively. Two hundred thirty-eight 238 developed IM. Five months after the diagnosis of IM, participants deemed not recovered and a number of matched, recovered controls were invited back for a comprehensive medical and psychiatric examination [24]. Fifty-five of the 238 students with IM met the criteria for ME/CFS 6 months later; 67 recovered students were chosen as matched controls [24]; for more details, see Jason et al [24].

Participants in the cohort for the present study included the 50 students who met criteria for ME/CFS six months following IM and 63 recovered controls, who at the 6 month post IM medical examination, had HR and BP recorded after being recumbent for 5 minutes and then after 1 minute of standing. One control and one participant with ME/CFS were missing systolic blood pressure data; one patient with ME/CFS was missing HR data. Seventeen of the 50 patients with ME/CFS six months following IM met criteria for S-ME/CFS. The patient’s chart was reviewed to verify the diagnosis (ME/CFS, S-ME/CFS or recovered based on the medical examination and confirming self-report information), for basic demographic information (age, sex), and to record the heart rate and blood pressure readings that were obtained during the routine physical examination, first after being recumbent for 5 minutes resting in the dark and then after 1 minute of standing. For the purposes of this study, postural tachycardia, (PT), as a component of IOH, was defined as a sustained increase in HR of at least 30 beats per minute when going from a supine to upright position in those 20-22 years of age and an increase in HR of at least 40 beats per minute in those 18-19 years of age [10-13]. IOH was defined as a decrease of at least 40 mm Hg in systolic BP or a decrease of at least 20 mm Hg in diastolic BP upon standing up for one minute [14,15]; however, a preliminary examination of our data did not reveal any BP changes of this magnitude, so we also examined a decrease of at least 20 mmHg in systolic BP or a decrease of at least 10 mmHg in diastolic BP upon standing up for one minute, as per OH criteria [10].

Chi square statistics were used to determine if there was a relationship between the presence of PT, IOH and the diagnosis of ME/CFS following IM where the N was > 5 in all groups; where the N was < 5 in some groups, Fisher’s exact test was used.

The study was approved by the Institutional Review Boards of all involved institutions.

Results

Tables 1-3 show the results of our analyses. As mentioned in the Methods section, a preliminary examination of our data did not reveal any changes in systolic BP of > 40 mm Hg (maximum was 32 mm Hg in a single participant) nor any changes in diastolic BP > 20 mm Hg (maximum was 18 mm Hg and 13 mm Hg in 2 different participants, neither of whom had the systolic BP change of 32 mm Hg). Therefore, all data in Tables 2 and 3 reflect OH BP criteria [10].

Participants with ME/CFS, S-ME/CFS, and recovered controls were compared. Students ranged in age from 18-23 years (median 20 years); there were 73 females and 40 males. There were no significant mean age differences between conditions: 18.8 (0.5) years for students with S-ME/CFS, 18.9 (0.9) years for students with ME/CFS and 18.7 (2.6) years for the recovered controls [24]. None of our patients had a positive Romberg test, which in some studies has been linked to OI [25].

Table 1 shows the relationship between participants with ME/CFS, S-ME/CFS and recovered controls with respect to PT. There was no relationship between participants with ME/CFS and PT compared with recovered controls (Fisher’s exact test: S-ME/CFS vs Control: p = 0.11; ME/CFS vs Control: p=0.42, ME/CFS and S-ME/CFS vs Control: p = 0.18; overall p = 0.17).

Table 1: Comparison of participants with ME/CFS, S-ME/CFS and Recovered Controls: ME/CFS and Postural Tachycardia (PT).

Recovered Control ME/CFS

S-ME/CFS
PT

6% (N=4)

 15% (N=5) 18% (N=3)

No PT

 94% (N=59) 85% (N=27)

82% (N=14)
Missing Data

 3% (N=1)

Total

 100% (N=63) 100% (N=33)

100% (N=17)

P values: Control vs ME/CFS – 0.16. Control vs S-ME/CFS – 0.16.

Table 2 shows the relationship between participants with ME/CFS, S-ME/CFS and Recovered Controls with respect to IOH (using OH BP criteria10). Again, there was no relationship between participants with ME/CFS having more IOH (using OH BP criteria10) than recovered controls (S-ME/CFS vs Control: x2 = 1.53, p = 0.22, ME/CFS vs Control: x2 = 0.38, p=054, ME/CFS and S-ME/CFS vs Control: x2 = 1.09, p = 0.3; overall x2 = 1.56, p = 0.46).

Table 2: Comparison of participants with ME/CFS, S-ME/CFS and Recovered Controls: ME/CFS and IOH (using OH BP cutoffs [10]).

Recovered Control ME/CFS

S-ME/CFS
IOH

14% (N=9)

 21% (N=7) 18% (N=3)

No IOH

 86% (N=54) 79% (N=26)

82% (N=14)
Total

 100% (N=63)

 100% (N=33)

100% (N=17)

P values: Control vs ME/CFS – 0.39 Control vs S-ME/CFS – 0.71

Table 3 shows the relationship between participants with ME/CFS, S-ME/CFS and Recovered Controls with respect to either PT and/or IOH (using OH BP criteria10). Again, there was no relationship between participants with ME/CFS having either PT or IOH (using OH BP criteria10) when compared with recovered controls (S-ME/CFS vs Control: x2 = 1.50, p = 0.22 ME/CFS vs Control: x2 = 1.02, p=31, S-ME/CFS and ME/CFS vs Control: x2 = 1.76, p = 0.19; overall x2 = 1.9, p = 0.39).

Table 3: Comparison of participants with ME/CFS, S-ME/CFS and Recovered Controls: ME/CFS and PT or IOH (using OH BP cutoffs [10]).

Recovered Control ME/CFS

S-ME/CFS
PT and/or IOH

21% (N=13)

 36% (N=12) 35% (N=6)

No PT or IOH

 79% (N=50) 64% (N=21)

65% (N=11)
Total

100% (N=63)

 100% (N=33)

100% (N=17)

P values: Control vs ME/CFS – 0.10 Control vs S-ME/CFS – 0.21

Discussion

In a well-studied population of college students six months following IM, we found no significant relationship between PT or IOH (even using the less stringent OH BP criteria 10) and ME/CFS following IM. If a patient had both ME/CFS and IOH, therapeutic maneuvers (e.g., static handgrip or lower body tensing) might be helpful in alleviating some symptoms [26,27].

Autonomic dysfunction is thought to play a role in the pathophysiology of OH and POTS [28,29], although autonomic complaints may not correlate with the presence of autonomic dysfunction on physical examination [30]. The relationship between ME/CFS and autonomic dysfunction has been seen in many [6-9,28,29,31,32] but not all 20,33 previous studies. There are studies that suggest that OI may characterize only a subgroup of those with ME/CFS [34,35], and the data presented here and previously [20] do not exclude that possibility. If present, the diagnosis of OI in patients with ME/CFS may provide some direction for management of the often debilitating symptoms of ME/CFS, such as increasing salt and fluid intake and the use of compression stockings and certain medications [28,29,36].

Strengths of our study include its prospective nature and the gathering of data before the final diagnosis (ME/CFS vs recovered control) was known, leading to unbiased data collection. The main limitation of our study is the lack of performing a 10 minute standing test of orthostatic intolerance, although we did this in a previous study in a similar population of patients with ME/CFS following IM [20] and found no relationship between the diagnosis of ME/CFS following IM and OI. Other limitations include the lack of tilt table testing and not examining patients on bad days, when OI may be more prominent [25].

Conclusions

In conclusion, we found no significant relationship between IOH and the diagnosis of ME/CFS in a well-studied prospective cohort of college students who developed ME/CFS six months following IM, as we found no relationship between abnormalities in orthostatic tolerance testing between a similar sample of participants with ME/CFSD following IM and recovered controls [20].

List of Abbreviations

BP: Blood Pressure; HR: Heart Rate; Ig: Immunoglobulin; IM: Infectious Mononucleosis; IOH: Initial Orthostatic Hypotension; ME/CFS: Myalgic Encephalomyelitis/Chronic Fatigue Syndrome; OH: Orthostatic Hypotension; OI: Orthostatic Intolerance; POTS: Postural Orthostatic Tachycardia Syndrome; PT: Postural Tachycardia; S-ME/CFS: Severe ME/CFS; VCA: Viral Capsid Antigen

Declarations

Ethics approval and consent to participate: The study was approved by the Institutional Review Boards of DePaul University (Protocol # LJ09031PSY-R36) and the Ann & Robert H Lurie Children’s Hospital of Chicago (IRB 2020-34867 in accordance with the Declaration of Helsinki. Written consent was obtained from all participants.

Consent for Publication

N/A

Availability of Data/Materials

Materials described in this manuscript, including all relevant (de-identified) raw data will be freely available to any researcher wishing to use them for non-commercial purposes, without breeching participant confidentiality. The datasets generated during and/or analyzed during the current study are available from the corresponding author on reasonable request.

Competing Interests

The authors report that there are no competing interests to declare.

Funding

Supported by the National Institute of Allergy and Infectious Diseases (grant number AI 105781) to Leonard A Jason and Ben Z Katz.

Authors’ Contributions

SS, ES, ML and JF made substantial contributions to the acquisition, analysis, and interpretation of data, have made substantial revisions to the work, have approved the submitted version and have agreed both to be personally accountable for their own contributions and to ensure that questions related to the accuracy or integrity of any part of the work, even ones in which they were not personally involved, are appropriately investigated, resolved, and the resolution documented in the literature.

LAJ made substantial contributions to the conception and design of the work, helped in the analysis and interpretation of the data, has made substantial revisions to the work, has approved the submitted version and agrees both to be personally accountable for his own contributions and ensures that questions related to the accuracy or integrity of any part of the work, even ones in which he was not personally involved, are appropriately investigated, resolved, and the resolution documented in the literature.

BZK made substantial contributions to the conception and design of the work, was involved with the acquisition, analysis, and interpretation of the data, drafted the first version of the work and then substantively revised it, has approved the submitted version and agrees both to be personally accountable for his own contributions and to ensure that questions related to the accuracy or integrity of any part of the work, even ones in which he was not personally involved, are appropriately investigated, resolved, and the resolution documented in the literature.

SS,ES, ML and JF made substantial contributions to the acquisition, analysis and interpretation of the data, have made substantial revisions to the work, have approved the submitted version and have agreed to be personally accountable for their own contributions and to ensure that questions related to the accuracy or integrity of any part of the work, even ones in which they were not personally involved are appropriately investigated, resolved and the resolution documented in the literature.

LAJ and BZK made substantial contributions to the conception and design of the work and helped in the acquisition, analysis and interpretation of the data. BZK drafted the first version of the work. LAJ and BZK have made substantial revisions to the work, have approved the submitted version, and agree to be personally accountable for their own contributions and to ensure that questions related to the accuracy or integrity of any part of the work, even ones in which they were not personally involved are appropriately investigated, resolved and the resolution documented in the literature.

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Navigating a Male-Dominated Discipline: Learning and Development of a Female Master’s Student in Pure Sciences in Taiwan

2 Replies

DOI: 10.31038/PSYJ.2025742

Abstract

This case report examines the learning and developmental experiences of “Ann,” a 24-year-old female master’s student and the sole woman in a theoretical physics program at a research-oriented university in Taiwan. Using a phenomenological qualitative approach, data were collected through semi-structured interviews and analyzed thematically. Four primary themes emerged: antecedent factors influencing her decision to pursue theoretical physics, experiences of collaboration with male peers, persistence in continuous learning, and factors facilitating her success in a male-dominated field. The report concludes with recommendations for policy and practice to support women in pure sciences, as well as directions for future research.

Keywords

Female graduate student, Pure sciences, Learning, Development, Competence

Introduction

In Taiwan, mathematics and science remain strongly male- dominated, with scientists frequently perceived as predominantly male. According to Ministry of Education statistics [1], women represent only 32.61% of graduate students in physics, chemistry, or earth science, and 31.83% in mathematics or statistics. Within pure sciences—such as pure mathematics, theoretical physics, and mathematical physics— female representation is even lower than in other STEM disciplines. This scarcity may discourage prospective female students from entering these fields, perpetuating gender disparities. Addressing this imbalance requires a deeper understanding of the lived experiences of women in pure sciences. This study focuses on “Ann,” who completed a bachelor’s degree in physics and is now pursuing a master’s degree in theoretical physics at a research-oriented university in Taiwan. Her coursework spans a broad range of pure science subjects. Here, “pure sciences” refers to disciplines grounded in theoretical principles, while “theories” denotes the concepts, principles, and knowledge specific to these fields.

Gender Roles and Higher Education in Taiwan

Traditional Taiwanese cultural norms have long emphasized female domestic responsibilities—childbearing, childcare, and elder care—alongside values of female submissiveness and male dominance [2]. However, shifts in women’s educational attainment, delayed marriage, and increased workforce participation have led to evolving gender roles [3]. Many female university students now seek autonomy, independence, and professional competence, yet still navigate tensions between traditional expectations and more egalitarian ideals [4]. Gender disparities in higher education persist: men are more likely to pursue STEM majors, while women are concentrated in the humanities [5,6]. Studies of Taiwanese STEM environments reveal that science and engineering laboratories often reflect masculine cultures, with hierarchical structures privileging male members and marginalizing women [7,8]. Female STEM graduates’ career choices are shaped by personal interest, academic performance, institutional prestige, family expectations, and significant life events [9], yet constrained by gender stereotypes, discrimination, and work–family conflicts [10].

Research on successful female scientists in Taiwan highlights qualities such as intellectual curiosity, perseverance, time management, and passion for research, supported by parental encouragement, mentorship, and professional opportunities [11]. Other studies emphasize the importance of interest-driven learning, supportive peer networks [12-14], diverse career values, and opportunities for creativity and problem-solving [15,16]. However, persistent gendered stereotypes in science and technology remain embedded in both cultural narratives and media representations [17,18].

Female STEM Students Worldwide

Globally, women in STEM face systemic challenges. Institutional climate, lack of social support, and feelings of alienation in male- dominated fields contribute to attrition from graduate programs [19]. A sense of belonging is crucial for sustaining engagement, yet studies show it often declines over time, particularly in engineering [20,21]. Barriers include male dominance, limited awareness of opportunities, the scarcity of female role models (FRMs) and mentors, heavy time demands, insufficient encouragement, and perceptions of a glass ceiling [22]. Support networks play a protective role. Social support from family, peers, and teachers is linked to more positive attitudes and stronger self-perceptions in STEM [23]. Female classmates and FRMs can reduce isolation, foster collaboration, and provide emotional support. The presence of female faculty improves satisfaction, career aspirations, and academic outcomes [24,25], while same-gender mentors enhance comfort in research settings for underrepresented students [26].

Persistent stereotypes about women’s ability in STEM—such as assumptions of weaker mathematical skill [27] or lesser innate talent [28]—erode self-efficacy [29] and scientific identity [30]. Female role models have been shown to counter these effects, improving performance, retention, and belonging [31,32].

Relationships, Connection, and Engagement

Relational-Cultural Theory [33,34] emphasizes that human growth occurs within relationships, and that women, in particular, are driven by the need for connection. Growth-fostering relationships are marked by mutual empathy and empowerment. Women tend to prioritize intimacy, trust, and mutual support in friendships, engaging in greater emotional self-disclosure and communication compared to men [35-38] .In Taiwan, academic involvement and peer relationships significantly shape university students’ psychosocial development [39,40]. Student engagement—defined as the degree of connection to meaningful academic and social activities—is a strong predictor of success [41-43]. Engagement reflects both the time and effort students invest and the institutional supports that encourage participation.

Rationale of the Study

Engagement is central to academic success, persistence, and satisfaction [44]. In STEM contexts, it encompasses the academic and social dimensions essential for retention [45] and is shaped by stereotypes, bias, campus climate, identity formation, and belonging [46]. This study applies Fredricks et al.’s [47] three-dimensional engagement framework: (1) behavioral engagement — effort, persistence, attendance, and constructive participation, (2) emotional engagement — positive and negative reactions toward peers, teachers, and academic content, reflecting belonging and identification, and (3) cognitive engagement — self-regulation, deep learning strategies, and sustained effort to master complex material. These dimensions are examined in the context of Ann’s higher education experience, focusing on instructor–student interaction, peer collaboration, and positive coping strategies within a science-supportive culture. This framework provides the lens through which her learning and development in a male-dominated pure sciences program are explored.

Method

This study adopted a phenomenological approach, which aims to provide detailed descriptions of individuals’ ordinary, lived experiences and to identify the essential structures of those experiences. This approach was used to explore and articulate the learning and developmental experiences of a female graduate student enrolled in a theoretical physics program in Taiwan.

Participant

The participant, referred to as Ann, is a 24-year-old master’s student at a research-oriented university in Taiwan, majoring in theoretical physics. She is the only female student in both her academic program and research team. Ann voluntarily participated in the study, sharing her reflections on her learning trajectory and personal development in the male-dominated field of pure sciences.

Interviewer

Data collection was conducted by a research assistant (RA) with a master’s degree in counseling. The RA had completed formal coursework in interviewing skills, qualitative research, and research methodology, as well as pilot studies to refine her interviewing competence. She established rapport with Ann prior to the interviews and maintained an open, nonjudgmental stance throughout the process.

Data Collection

Two in-depth interviews were conducted, each lasting between 90 and 120 minutes. Prior to participation, Ann was fully informed about the study’s purpose and procedures and provided written informed consent. The interviews were designed to elicit rich, detailed narratives of her experiences. Sample guiding questions included: “Please describe your learning and development experiences in the field of pure sciences.” “Please share any significant or memorable perceptions and reactions you have had during your academic career in pure sciences at the higher education level.” “If applicable, what advice would you offer to prospective female students considering enrollment in a pure sciences program?”

Data Analysis

The author conducted the data analysis following Creswell’s [48] phenomenological procedures. To minimize bias, the researcher engaged in bracketing, setting aside personal assumptions about female students in pure sciences to focus on uncovering new and fundamental meanings from the data. The analytic process included the following steps: (1) reviewing transcripts, scanning materials, typing field notes, and organizing data from multiple sources, (2) reading the entire dataset for a holistic understanding, reflecting on overarching meanings, and recording general impressions, (3) dividing the text into meaningful segments and applying initial codes, (4) developing detailed descriptions of the participant and context, grouping codes into preliminary themes, (5) refining themes through iterative analysis to construct a comprehensive thematic framework, (6) building complex connections among themes and integrating them into an overarching narrative, and (7) formulating a final description that captures the essence of the participant’s lived experience.Several strategies were employed to enhance the study’s validity and reliability. A detailed research protocol and database were maintained [49]. Transcripts were checked for accuracy, and codes were continuously compared with the raw data. Validation strategies recommended by Creswell and Miller [50] were used, including prolonged engagement, persistent observation, and triangulation of multiple sources and methods. Rich, thick descriptions were produced to convey contextual detail. Member checking was conducted by inviting Ann to review preliminary drafts and provide feedback. Finally, a peer external to the research team served as an auditor, reviewing both the research process and the findings for consistency and rigor.

Results

Antecedent Factors for Learning

Intrinsic Motivation: Learning Science to Understand the World

Since her senior year of high school, Ann has been deeply motivated by a desire to understand the world through the study of pure science theories. She explained, “As long as students enter the field of pure sciences, they have a dream to explore and understand the world. I belong to this group, and I desire to learn theories to reach this goal.”

Academic Goal: Enhancing Professional Competence

As the only female graduate student in her theoretical physics research team, Ann seeks to strengthen her academic abilities and establish herself as a competent professional. She enrolled in the program to prove her capability in mastering pure sciences, aspiring to perform at a level equal to or higher than her male peers: “I am the only female in our entire research team… I want to encourage young women to join this program. The first thing is to make myself better, even better than my male peers. I work hard to be a competent student who stands on my own feet!”

Personality Traits: Persistence, Patience, and Flexibility

Ann describes herself as introverted, autonomous, and independent, qualities she believes are well-suited for studying pure sciences. She emphasizes her persistence, tolerance for solitude, and ability to adapt in challenging learning environments: “I am less afraid of being alone, and I am less likely to give up because of difficulties in learning… My introverted personality makes me quite suitable for studying pure sciences.” She further notes that, unlike many female students who may feel isolated in male-dominated programs, she has prepared herself to manage such solitude: “Few or no female companions in the program make it unbearable for most female students, not for me. As the only female student in pure sciences, I mainly count on myself.”

Resilience and Independence in a Gender-Imbalanced Environment

Ann acknowledges that many female STEM students prefer programs with greater female representation, partly to avoid feelings of isolation. She believes that women considering pure sciences must develop resilience, problem-solving skills, and a capacity for independent thought: “I prepare myself to be independent, enhance my problem-solving abilities to manage my studies and life, and tolerate solitude.”

Navigating Collaboration with Male Peers

Collaborative Learning as a Strategy to Overcome Barriers

Given the scarcity of female students in pure sciences, Ann relies on male peers for academic discussions, which she considers essential for mastering complex theoretical concepts: “Because academic discussions occur frequently among men, they gather and discuss theories anytime, anywhere… Without academic discussions, it is difficult for female students to learn theories in pure sciences.”

Maintaining Professional Boundaries

While engaging in collaborative learning, Ann is deliberate about maintaining clear professional boundaries to avoid misunderstandings or unwanted social complications: “I believe that there must be a clear boundary between males and females… I have to maintain a clear boundary to demonstrate that we are partners in learning.”

Persistence and Long-Term Learning Strategies

Steady Effort in a Challenging Field

Ann views learning theoretical physics as an inherently slow and incremental process. Despite the difficulty, she remains committed to steady progress: “If I learn theories, I won’t necessarily achieve significant outcomes soon… studying theories takes time and proceeds step by step with patience.”

Preparation and Focused Discussions

Before approaching peers with questions, Ann engages in independent literature review and problem analysis: “If I want to discuss something… I first think about it carefully and check with the relevant literature to gain a certain degree of understanding.”

Gradual Development of Self-Reliance

Over time, Ann has cultivated enough expertise to work more independently. Being appointed as a teaching assistant was a milestone that reinforced her confidence and self-efficacy: “When I’m good enough… I don’t need to depend on others much… I don’t feel like being the only female in this team is bad anymore.”

Factors Promoting Learning

Female Role Models

Ann underscores the motivational influence of female scholars, teachers, and senior students in pure sciences. These role models inspire her through their dedication and passion: “I met a senior female graduate student who had a great impact on me… She, majoring in pure mathematics, claims that she is unique because a majority of people in the world cannot understand what she is studying.”

Female Peer Networks

Although she lacks female peers in her immediate research group, Ann builds friendships with women from other departments, valuing emotional support and shared experiences: “Women can establish close friendships… and better support each other in daily lives.”

Career Orientation and Patience for Non-Mainstream Fields

Ann acknowledges that pure sciences research is often more aligned with cutting-edge exploration than with popular industry applications. She accepts that her career path may diverge from mainstream STEM employment trends: “We are usually doing more cutting-edge research, not like semiconductors… that are currently popular in Taiwan.”

Interpersonal Adaptability

Recognizing that many in her field are introverted or self-focused, Ann actively adapts her interpersonal approach to work effectively with colleagues of varying personalities: “To survive in pure sciences, I have to be brave enough to get along and work with these men… with different characteristics.”

Family Support

Ann credits her family’s encouragement and lack of financial pressure as key enablers of her academic persistence: “My parents respect my personal interest and decision… My family did not push me to find a job soon, did not blame me for majoring in pure sciences because of its non-mainstream position in the job market.”

Discussion

Awareness of Gender Stereotypes

Ann is acutely aware of gender stereotypes in mathematics and science, particularly the perception that pure sciences—especially theoretical disciplines—are male domains. Such stereotypes, which assume that men have greater aptitude for theoretical work, discourage many young women from enrolling in STEM majors. Ann’s awareness echoes findings that male-dominated images of engineers persist in Taiwan and that the gender structure in Taiwan’s science and technology fields remains unfavorable to women.

She also observes the scarcity of female classmates in pure sciences, which reinforces the field’s lack of appeal for women and perpetuates the cycle of underrepresentation. This reflects literature linking gender stereotypes to the association of men with science and women with non-science in Taiwan [51]. While pure sciences are male-dominated and masculinity-oriented, Ann appears largely unaffected by these stereotypes. She adopts coping strategies to overcome learning obstacles, improve performance, and maintain progress. One of her motivations is to prove that women can succeed in pure sciences, a finding consistent with research on Taiwanese female mechanical engineering students who aimed to counter gender stereotypes through achievement.Although she faces barriers common to women in STEM—male dominance, limited awareness of opportunities, and a lack of female role models and mentors —Ann demonstrates resilience and problem-solving ability. This contrasts with Liu’s finding that negative stereotypes lower self-efficacy for female STEM graduates in Taiwan. Ann’s experience is atypical, differing from studies that describe women’s inferior status in STEM due to combined internal and external barriers such as low self-efficacy, limited support, and gender-biased environments.

Personal Interests and Personality Characteristics

Ann’s decision to specialize in pure sciences stems from her personal interest, career goals, and motivation to pursue knowledge, alongside support from parents, female teachers, and peers. This aligns with research showing the influence of personal interests [52] and high motivation in STEM career Recognizing her aptitude for theoretical study, Ann is driven by passion, ambition, and a commitment to becoming a scholar.Her perseverance, independence, courage, resilience, and persistence align with Hsu’s [53] observation that women in nontraditional fields often display these Ann’s time-management skills, professional growth, and steadfastness reflect characteristics of outstanding Taiwanese female scientists and technologists. She approaches problems with curiosity and adaptability, consistent with Sung and Kao’s findings on positive traits among Taiwanese female STEM undergraduates. Ann’s clear academic goals, combined with her willingness to endure solitude and sustain effort, help her overcome gender stereotypes and barriers in male-dominated environments. This mirrors Swafford and Anderson’s conclusion that persistence and personal expectations are key for women in STEM, and further contrasts with Liu’s view that stereotypes lead to internal barriers.

Connections with and Social Support from FRMs and Female Peers

Ann values friendships with female peers, especially in dormitories and clubs, for emotional support and connection. This aligns with research showing that women value supportive, cooperative learning relationships [54]. Supportive peer and faculty relationships have been shown to influence female engineering students’ career choices. She notes that the absence of female companionship can lead women to drop out of STEM programs, echoing concerns about inadequate mentorship and social support. Interactions with female peers proficient in mathematics can boost identification with the subject and persistence. Ann’s emphasis on meaningful relationships and emotional intimacy aligns with findings that women place high value on connection and supportive growth.Ann also recognizes the importance of female role models (FRMs) in providing support and enhancing belonging in STEM [55]. However, due to the scarcity of FRMs in pure sciences, she actively seeks female mentors and peers through conferences and external academic activities. Such connections reinforce her confidence and identity as a scientist.

Collaboration with and Adjustment to Male Peers

In a male-dominated field, Ann collaborates with male peers for problem-solving while maintaining professional boundaries to avoid misunderstandings. She acknowledges that the abstract nature of theoretical work requires continuous discussion, and that engaging with male peers is often necessary due to the scarcity of women in her field. This partially reflects Fairlie’s [56] finding that female–male partnerships in STEM do not necessarily hinder academic outcomes. However, Ann also notes that men more easily navigate resources and systems in masculine academic cultures, reinforcing her need for female peer and mentor connections. The findings are consistent with Han’s assertions, which highlight how hierarchical structures tend to privilege male members while systematically marginalizing women.

Support from Parents and Family Members

Ann emphasizes the importance of family support in her decision to pursue pure sciences. Free from immediate financial pressure due to her family’s backing, she can focus on long-term academic and career goals. This supports prior findings on the positive impact of parental encouragement on women entering STEM or nontraditional fields. Her experience contrasts with Liu’s and Hung’s [57] findings on women prioritizing family over career or abandoning aspirations due to economic pressures, and aligns with Rice et al.’s findings on the benefits of broad social support.

Engaged Learner and Professional Identity Building

Despite her awareness of gender bias, Ann remains committed to her goal of becoming a pure scientist. Her dedication reflects trends in Taiwanese women increasingly rejecting traditional gender roles. Ann shows strong behavioral engagement (attendance, effort, persistence), cognitive engagement (self-regulation, group discussions), and emotional engagement (positive attitudes toward peers, faculty, and her academic environment). This multidimensional engagement fosters her academic success and sense of belonging, consistent with Fredricks et al. and Kuh et al..Through her participation in the pure sciences community, Ann develops professional pride and identity, equipping herself with both academic competence and career readiness. This reinforces the importance of personal and career interest, recognition of career pathways, and self-awareness in women’s choice of nontraditional fields [58].

Implications and Conclusions

To attract and retain more women in pure sciences, universities should actively recruit female professors, professionals, and students, and provide resources such as mentorship programs. Counseling and educational initiatives should counter gender stereotypes and cultivate supportive institutional climates. Activities that foster interpersonal support and social networks are essential. The presence of female peers and FRMs can inspire persistence, while male peers can contribute by facilitating integration into study groups and research teams.

Acknowledgements

This study is a part of a project. It was sponsored by a grant (MOST 108-2629-H-007-001) from the National Science and Technology Council, Taiwan.

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