Circadian regulation is increasingly recognized as a critical determinant of tumor biology, immune dynamics, and therapeutic response. However, standardized and reproducible laboratory methodologies for assessing circadian-related biomarkers remain limited in human tumor tissues in general, and in translational and clinical research settings in particular. Here, we described a pragmatic and reproducible protocol for circadian gene expression profiling in formalin-fixed paraffin-embedded (FFPE) solid tumor samples, focusing on the core clock genes PER2 and BMAL1, together with the clinically relevant resistance marker AR-V7. The workflow integrates mandatory real-time time-stamping at biopsy acquisition, standardized pre-analytical quality control parameters, and parallel assessment of protein- and transcript-level expression from matched FFPE sections. Immunohistochemistry was performed using analytically validated antibodies on automated staining platforms and quantified through a combination of semi-quantitative scoring and digital image analysis using open-source pathology software. In parallel, RNA was extracted from adjacent FFPE sections and analyzed by RT-qPCR with standardized primer sets and normalization to reference genes (ACTB, GAPDH, and RPLP0). Additional quality-control measures included antibody validation and lot-to-lot consistency checks, automated scoring pipelines, RNA integrity assessment, and evaluation of intra- and inter-operator variability. The protocol was designed to enable robust assessment of analytical feasibility, reproducibility, and concordance between protein- and transcript-level measurements, while allowing exploratory correlation with clinical variables such as PD-L1 expression, systemic inflammatory indices, and timing of systemic therapy. This circadian-aware workflow provides a scalable methodological foundation for integrating temporal biology into precision oncological research and supports future multicenter validation and chrono-therapeutic studies.
Background: Amid global health concerns, extended-spectrum beta-lactamase (ESBL)-producing bacteria create substantial resistance challenges to current antibiotics. ESBL-producing pathogens are resistant to many antibiotics, including first- to third-generation cephalosporins, monobactams, or other antibiotic classes. Therefore, healthcare relies on carbapenems and colistin as their last options. Alarmingly, resistance to these critical drugs is increasing, emphasizing the urgent need for contemporary solutions, such as phage therapy. Objective:To develop an integrated workflow for identification and phage susceptibility assessment of ESBL-producing E. coli from environmental water samples. Methods: In this study, 15 downstream water samples were collected from five locations in Islamabad and Rawalpindi, including Nala Lai, Chattar Park, Shahdara, Nescom Hospital H-10, and G-10. Results: Of these, 10 samples (66.67%) tested positive for Escherichia coli, while five (33.33%) were negative. All samples from Chattar Park were negative for E. coli. Screening via the double-disk synergy test and combination disk test identified four samples (26.66%) with ESBL-producing E. coli and six (40%) with non-ESBL-producing isolates. Bacteriophages were isolated from Nescom Hospital stream water using the double agar overlay method and purified with a 0.45 μm syringe filter. The phages exhibited lytic activity against ESBL-producing E. coli NLA-3, as indicated in spot test results, and their therapeutic potential was validated through reduction assays. The isolated phage NHE-1 shows promise as an alternative treatment for antibiotic-resistant infections. Conclusion: Further studies are needed to expand its application to diverse bacterial strains and evaluate its clinical efficacy. Utilizing bacteriophages’ natural ability to target bacteria may provide sustainable, effective solutions to combat antibiotic resistance.
Background: Polycystic ovary syndrome (PCOS) represents the most common endocrine disorder afflicting women of reproductive age. PCOS is characterized by hormonal imbalances, metabolic abnormalities, and infertility, yet only limited therapeutic options are available. Metformin and Bushen Ditan Decoction (BDD), a traditional Chinese medicine formula, are commonly used in Western and traditional Chinese medicine, respectively, for treating PCOS. Despite their demonstrated clinical efficacy, the mechanisms underlying their therapeutic effects remain incompletely understood. Objective: This study aimed to investigate the therapeutic mechanisms of metformin and BDD in a PCOS rat model. Methods: PCOS was induced in rats by using the Poretsky method. After model induction, the animals were given metformin (150 mg/kg/day) or BDD (8, 16, or 32 mg/kg/day) via intragastric gavage for 4 weeks. Results: Both metformin and BDD significantly reduced testosterone levels and improved lipid metabolism parameters. MicroRNA (miRNA) sequencing of ovarian tissues identified 18 miRNAs commonly regulated by both treatments, and functional enrichment analysis indicated that these differentially expressed miRNAs are involved in autophagy- and metabolism-related pathways implicated in the pathogenesis of PCOS. Conservation analysis across species highlighted the potential clinical relevance of these findings. Conclusion: This study demonstrated how mechanistically metformin and BDD work on PCOS, particularly through the regulation of autophagy- and metabolism-related pathways, providing insights for their future clinical applications.
Background: Soluble urokinase plasminogen activator receptor (suPAR) reflects immune activation and chronic inflammatory responses and has been proposed as a prognostic biomarker in several acute conditions. Objectives: This study aims to evaluate the clinical performance of two analytical methods for plasma suPAR determination in patients with stroke: a point-of-care (POC) lateral-flow assay and an automated particle-enriched turbidimetric immunoassay performed on a biochemistry analyzer. Methods: Plasma samples from 27 patients with stroke and 14 age-matched healthy controls were analyzed with both methods. suPAR concentrations were significantly higher in stroke patients compared with controls for both assays. Results: A statistically significant difference between POC and analyzer measurements was observed only within the stroke group (p=0.024), and a strong positive correlation between the two methods was identified (r = 0.745, p<0.001). Receiver operating characteristic analysis demonstrated higher sensitivity for the POC method (96%) compared with the analyzer (82%), while the specificity was identical (57%). Conclusion: These findings suggest that suPAR measurement using a rapid POC platform provides reliable results and may support early risk stratification in patients with stroke in emergency department settings.
Background: Infections caused by multidrug-resistant organisms pose a significant global health challenge due to the limited therapeutic options available. Synergistic drug combinations are often employed to enhance treatment efficacy. Because peptidoglycan is unique to prokaryotes, many studies focus on combinations that include beta-lactam antibiotics. To address bacterial strains that produce beta-lactamases, a range of beta-lactamase inhibitors has been developed. The European Committee on Antimicrobial Susceptibility Testing (EUCAST) and the Clinical and Laboratory Standards Institute (CLSI) regularly update clinical guidelines to include standards for new beta-lactam/beta-lactamase inhibitor combinations. Objective: This work provides an overview of beta-lactam and beta-lactamase inhibitor combinations, together with updated clinical guidelines and an analysis of the relevant Protein Data Bank (PDB) entries related to these synergistic combinations. First, the latest EUCAST and CLSI guidelines are reviewed to identify the most recent beta-lactam/beta-lactamase inhibitor combinations used in therapy. Next, PDB entries related to these combinations are examined and compared with new compounds under investigation. EUCAST and CLSI guidelines are valuable for guiding therapeutic strategies, not only for interpreting antibiograms. The newest beta-lactamase inhibitors include avibactam, relebactam, vaborbactam, and enmetazobactam. X-ray crystallography studies have enhanced understanding of inhibitor interactions with major beta-lactamases in Gram-negative pathogens—serine beta-lactamases and metallo-beta-lactamases. Conclusion: Investigating synergistic combinations of beta-lactams and beta-lactamase inhibitors is promising because beta-lactams target peptidoglycan and may enable new therapeutic strategies.
