Carnosine and anserine are bioactive, histidine-containing dipeptides recognized for their antioxidant, pH-buffering, and neuroprotective functions. However, their precise quantification is frequently compromised by their high structural similarity and complex matrix-induced analytical interferences. This study developed and validated an optimized, simplified extraction protocol coupled with high-performance liquid chromatography diode array detection (HPLC-DAD) for the simultaneous determination of both dipeptides across diverse meat matrices, including Thai native black-bone chicken (Nin-Kaset), commercial broiler, pork, beef, and buffalo meat. Extraction efficiency was systematically optimized by modulating the solution pH. Under optimized conditions, the validated method demonstrated excellent linearity (1–200 µg mL−1, r2 ≥ 0.9990) with limits of detection and quantification (LOD/LOQ) values at 0.0093/0.0283 mg g−1 for carnosine and 0.0139/0.0420 mg g−1 for anserine. Method accuracy was confirmed by satisfactory recoveries ranging from 96.64% to 102.68%, with repeatability values (RSDs) of 0.70–14.18%. Application to real matrices revealed distinct dipeptide profiles. Pork contained the highest carnosine level (3.59 ± 0.19 mg g−1), whereas white-feather Nin-Kaset exhibited the highest anserine level (6.77 ± 0.40 mg g−1). Overall, the validated framework offers a rapid, cost-effective, and robust approach for routine dipeptide profiling, supporting future applications in food authentication, nutritional evaluation and functional food development.
Epidermal development and regeneration rely on the stepwise differentiation of keratinocytes, the primary constituent cells of the epidermis. In the integrated epidermis, keratinocyte differentiation (KD) occurs in response to the increasing calcium gradient. Reflecting this, high-calcium incubation remains a popular approach for inducing KD in vitro. The outcome is primarily assessed by differential expression of differentiation markers compared to proliferative cells. However, it is significantly influenced by the cell models used, calcium concentration and incubation time, which often vary between studies. Such differences compromise reproducibility and comparability. In this article, we overview major parameters that shape the outcome of high-calcium induced KD. Comparative experimental data between high-calcium incubation and high-density induced differentiation underscore the need for combining additional parameters, such as microscopy based visualization with marker protein analysis for unbiased assessment of KD. These recommendations are intended to guide future research in keratinocyte biology.
Background: The human amniotic membrane (hAM) is widely used in tissue engineering due to its low immunogenicity and extracellular matrix (ECM) rich in structural proteins; in this context, decellularization aims to remove cellular components while preserving tissue integrity, and this study aimed to compare the effects of two enzymatic methods (trypsin and thermolysin). Methods: Human amniotic membranes obtained from placentas were divided into three groups: non-decellularized control, trypsin-treated, and thermolysin-treated. The membranes were treated with 0.25 trypsin-EDTA for 60 min at 37 °C, followed by incubation with Triton X-100 for 30 min at 37 °C, or with 125 µg/mL thermolysin for 9 min at 37 °C. Histological analysis with hematoxylin–eosin and semi-quantitative scoring assessed epithelial and stromal integrity, immunohistochemistry evaluated laminin α and fibronectin expression with quantitative image analysis, and uniaxial tensile testing measured mechanical properties; no preregistration numbers or animal models were reported. Results: Trypsin achieved more effective epithelial removal, whereas thermolysin caused greater stromal disruption; no statistically significant differences in laminin α or fibronectin expression were found among groups (p > 0.05), indicating preservation of key ECM components; both treatments reduced mechanical performance, with thermolysin decreasing early- and mid-path stiffness and trypsin reducing late-path stress and work density. Conclusion: Both enzymatic protocols preserved ECM composition but induced distinct structural and mechanical alterations, suggesting that method selection should balance efficient cell removal with preservation of structural and functional properties according to the intended application.
A comprehensive assessment of allograft quality is essential for predicting successful transplantation and graft survival. Elevated levels of flavin mononucleotide (FMN) in perfusate obtained during hypothermic oxygenated perfusion (HOPE) may serve as a promising predictor of early graft dysfunction and post-transplant complications. The aim of this study was to evaluate the feasibility of applying an FMN determination method to HTK-based perfusion solutions used during liver machine perfusion. The spectrofluorimetric method for FMN determination in modified HTK perfusion solution (modHTK) was evaluated for the following parameters: linearity, accuracy, repeatability, reproducibility, and stability. The study demonstrated the suitability of the quantitative FMN determination method for modHTK solution used during HOPE of liver grafts.
While the recently developed tissue clearing protocols pathoDISCO and activeDISCO significantly accelerate the clearing of large tissue specimens through active chemical dehydration using 2,2-dimethoxypropane, the final step of refractive index (RI) matching with viscous organic solvents as dibenzyl ether (DBE) remains restricted by slow passive diffusion. To overcome this bottleneck, we applied 40 kHz ultrasound using a standard, cost-effective laboratory bath to significantly enhance the diffusion kinetics of the clearing medium into large specimens. Our investigation on multi-centimeter-sized porcine muscle and human earlobe samples demonstrates that 40 kHz acoustic oscillations generated by a standard ultrasound cleaning device not only accelerate the clearing process but also yield superior and stable long-term optical transparency. We also tested 1 MHz high-frequency ultrasound but it offered no kinetic advantages and tended to induce tissue micro-fractures, an artifact we have not observed at 40 kHz at comparable energy levels. We therefore propose that standard 40 kHz ultrasound baths, ubiquitous in laboratories for cleaning purposes, represent an ideal and accessible tool for optimizing solvent-based tissue clearing.
Pancreatic ductal adenocarcinoma (PDAC) remains a highly lethal malignancy, highlighting the need for reproducible experimental systems that model pancreatic injury and subsequent neoplastic progression. Adult-onset genetically engineered models in which oncogenic Kras and mutant Trp53 are induced in the pancreas provide a relevant platform for studying PDAC pathogenesis, but efficient neoplastic progression often requires concomitant pancreatic injury. Cerulein-induced pancreatitis is widely used to provide an injury-associated inflammatory stimulus that promotes PDAC progression; however, repeated-injection regimens are labor-intensive and increase cumulative handling stress in animals. Here, we describe a dose-optimized, single-dose L-Lysine protocol designed to induce acute pancreatitis and characterize subsequent pancreatic neoplastic progression in adult-onset Kras/Trp53-driven mice. Tamoxifen-treated Ptf1aCreER/+; KrasLSL-G12D/+; Trp53LSL-R172H/+; Rosa26-RFP mice received a single intraperitoneal injection of L-Lysine, followed by biochemical, histological, and immunohistochemical assessment of pancreatic injury and tumor development. A single 2.0 g/kg L-Lysine injection induced sublethal acute pancreatitis characterized by increased serum pancreatic enzymes, interstitial edema, inflammatory cell infiltration, acinar cell necrosis, and rapid mitochondrial alterations. During 3–6 months of follow-up, mice developed multifocal acinar-to-ductal metaplasia, PanIN lesions, invasive PDAC, and peritoneal dissemination. This protocol provides a simple, synchronized, and less labor-intensive model for studying pancreatic injury and subsequent neoplastic progression in adult-onset Kras/Trp53-driven mice.
Natural coagulants have emerged as potential alternatives to synthetic chemicals in water treatment, especially for decentralized and low-resource applications. However, many previously reported Aloe vera-based coagulant preparation methods rely on drying, powder production, distilled water extraction, refrigeration, or other laboratory-dependent procedures that increase operational complexity and limit practical implementation. This study presents a simplified and rapid protocol for producing an Aloe vera-based natural coagulant using accessible materials and simplified preparation steps. The proposed methodology consists of extracting Aloe vera g13el, homogenizing 2 g of fresh gel with 50 mL of tap water using a household blender, and applying simple paper filtration to obtain the liquid coagulant. The protocol can be completed in less than 10 min without specialized laboratory infrastructure, energy-intensive processing, or laboratory-grade reagents. Coagulation performance was evaluated using synthetic turbid water with initial turbidity levels of 100, 200, and 300 NTU. Significant turbidity reduction was observed under all tested conditions, with several samples reaching residual turbidity values close to or equal to 0 NTU after 50-60 min of sedimentation. The results demonstrate the potential of the proposed protocol as a rapid, reproducible, and accessible approach for future investigation in point-of-use and decentralized water treatment applications.
Daphnids, commonly known as water fleas, are freshwater planktonic microcrustacean species used as model organisms in ecotoxicology, particularly in regulatory frameworks that adhere to OECD and ISO standards. Mortality is the most common endpoint in toxicity testing; however, more sensitive indicators are required to assess sublethal acute effects of pollutants. The use of feeding impairment as a toxicity phenotypic endpoint in daphnids is considered a cost-effective approach that aligns with the 3Rs principle (Replace, Reduce, Refine) and is more physiologically and environmentally relevant. Current feeding methods are inefficient due to the large test volumes and extended incubation periods required. In this paper, we present a miniaturised protocol to assess feeding behaviour following exposure to chemicals in daphnids. The method is based on the consumption of algae, which is measured with chlorophyll fluorescence. The optimised protocol is more robust and rapid, and results can be obtained in 30 min and in a 96-well plate. Responses in feeding rate were investigated using this miniaturised protocol following exposure to a range of prevalent pollutants, which include two metals and, as a more realistic sample, a leachate from smoked cigarette filters. All three pollutants were tested at sublethal concentrations. This method provides an efficient approach to assess the toxicity of chemicals and water quality.
Ensuring viral safety is a critical aspect of biopharmaceutical production, requiring sensitive and reliable methods for detecting adventitious agents. In this study, we systematically evaluated the performance of selected cell line–virus combinations to identify optimal models for in vitro viral detection assays. Three cell lines (Vero, MRC-5 and BHK-21 [C-13]) and representative model viruses (Reovirus type 3, Adenovirus type 5, Human parainfluenza virus type 3, and Herpes simplex virus) were analyzed in terms of cytopathic effect (CPE) kinetics, morphology, and detection sensitivity. All tested systems demonstrated high analytical sensitivity, with limits of quantification (LOQ) reaching 0.01 TCID50/mL for selected viruses. However, substantial differences were observed in infection dynamics and CPE morphology depending on the cell line–virus combination. BHK-21 [C-13] cells exhibited the most rapid and pronounced CPE for Reovirus type 3, enabling early and unambiguous detection. Vero cells provided robust and reproducible detection of Adenovirus type 5, characterized by well-defined cytopathic progression. MRC-5 cells showed controlled and consistent infection kinetics for both Human parainfluenza virus type 3 and Herpes simplex virus, allowing improved temporal resolution and interpretability. These findings demonstrate that assay performance depends not only on sensitivity but also on the kinetics and morphology of infection. Based on combined evaluation criteria, the following optimal cell line–virus pairs were identified: BHK-21 [C-13]/Reovirus type 3, Vero/Adenovirus type 5, and MRC-5/Human parainfluenza virus type 3 and Herpes simplex virus. The proposed approach supports rational selection of detection models and provides a preliminary descriptive framework for the development of routine visual screening assays in biopharmaceutical quality control.
Background: Small animal models are essential for atrial fibrillation (AF) research. Researchers in AF use an electrocardiogram (ECG), echocardiography and invasive electrophysiology study (EPS) to assess atrial structural and electrical remodeling. In relatively smaller cardiac structures and rapid heart rates, the examination can be challenging without special tools designed for animal study. Moreover, conventional invasive EPSs often cause significant trauma, alter autonomic tone, and limit longitudinal evaluations. This study aimed to evaluate the feasibility of repurposing hospital-grade medical devices for the non-invasive, multi-modality assessment of atrial myopathy in a rat model of metabolic syndrome (MetS). Methods: A total of 12 male Sprague-Dawley rats underwent the multi-modality assessment. Structural remodeling was evaluated using hospital-grade echocardiography (8-12 MHz) to measure left atrial (LA) dimensions and volume. Surface ECG was used to determine P-wave duration. Electrical remodeling and AF inducibility were assessed using transesophageal pacing (TEP)-based EPS, evaluating the atrial effective refractory period (AERP), sinus node recovery time (SNRT), and response to rapid atrial burst pacing. Results: The protocols showed high procedural safety (survival rate 91.67%) and successfully characterized atrial myopathy. Surface ECG showed marked intra-atrial conduction delay with prolonged P-wave duration in the MetS group (30.17 ± 4.62 vs. 22.33 ± 1.86 ms, p < 0.05). Echocardiography revealed signs of structural remodeling in the MetS group, evidenced by marked prolonged Isovolumic Relaxation Time (IVRT: 35.602 ± 3.043 vs. 19.187 ± 3.631 ms; p < 0.001) and increased Left Atrial Area (0.223 ± 0.0556 vs. 0.134 ± 0.033; p = 0.007). Furthermore, TEP-based EPS quantified electrical remodeling. The MetS group had shorter AERP (73.33 ± 10.33 ms vs. 120.00 ± 34.06 ms; p = 0.010) and Corrected SNRT (100.67 ± 53.98 ms) versus controls (208.33 ± 76.97 ms; p = 0.018). The MetS group exhibited a higher absolute AF inducibility rate (50%, three out of six rats) compared to the SH group (33.3%, two out of six rats). Conclusions: The integration of surface ECG, echocardiography, and TEP-based EPS provides a safe, highly reproducible, and comprehensive method for evaluating both structural and electrical components of atrial myopathy in small animal models, allowing for robust longitudinal studies.
(1) Background: Previous school-based interventions have addressed adolescent health behaviors such as physical activity, screen time, and sleep, but have predominantly targeted these behaviors independently rather than simultaneously. The Erasmus+ project FiTeens developed an integrated intervention combining theoretical content, videos, infographics, and interactive tasks to promote multiple health behaviors concurrently. The objective of the current article is to present the protocol for a school-based intervention study designed to examine the effects of the FiTeens program on adolescents’ physical activity, screen time, and sleep behaviors. We hypothesize that students receiving the FiTeens intervention will demonstrate increased physical activity, reduced screen time, and improved sleep outcomes compared with students in the control group. (2) Methods: Teachers will be introduced to the FiTeens tools prior to delivering the intervention to students in grades 5–9. Students will participate in an eight-week intervention program combining structured lessons and behavior-change challenges. Primary outcomes include changes in physical activity, screen time, and sleep duration and quality. Secondary outcomes include psychological determinants such as motivation and behavioral intentions. Data will be collected at baseline and at 1-, 3-, and 6-month follow-ups and analyzed using repeated measures ANOVA. (3) Expected results: The study will evaluate whether the intervention may contribute to improvements in health-related behaviors among adolescents, including increased physical activity, reduced screen time, and improved sleep outcomes. (4) Conclusions: The intervention based on FiTeens tools could have the potential to promote healthier lifestyle behaviors among students by increasing physical activity during leisure time, supporting the effective limitation of screen time and enhancing bedtime routines to improve sleep quality.
Background: This study focuses on developing a model of a non-healing wound that recapitulates the pathogenesis of the corresponding human pathology. Methods: A non-healing wound was modeled in mice with streptozotocin-induced diabetes. The following parameters were assessed: re-epithelialization, epidermal hypertrophy, wound contraction, relief index, angiogenesis, and granulation tissue maturation. These parameters were compared between diabetic mice and healthy controls. Results: The proposed model demonstrated a significant delay in regenerative processes compared to healthy animals. Conclusions: These findings support the relevance of this model to human pathology and indicate that it may be applicable for preclinical studies of drugs aimed at promoting wound regeneration.
The pathogenic free-living amoeba Naegleria fowleri is the cause of primary amebic meningoencephalitis (PAM), a central nervous system infection that is almost always lethal. One of the unusual features of the amoebae is the presence of ~4000 copies of a nucleolar-localized closed circular extrachromosomal ribosomal DNA element (CERE) that encodes the cell's ribosomal RNA repertoire. It has historically been challenging to purify large quantities of CERE, limiting our understanding of the nucleic acid. Here, we describe a methodology for CERE purification that improves yield, reduces processing times, and maintains the integrity of the plasmid. This approach will enable the study of this unique DNA architecture, advancing our understanding of the pathobiology of the organism.
The development and validation of a sensitive, rapid, and specific gas chromatography method for the evaluation of 19 common Class 2 and Class 3 solvents frequently used in nanomedicine formulation is described. Method validation was performed using PerkinElmer’s headspace gas chromatograph system with flame ionization detection and an Elite 624 Crossbond 6% cyanopropylphenyl-94% dimethylpolysiloxane or DB-Fatwax-Ultra Inert column with helium as the carrier gas. Validation characteristics such as linearity, spike recovery, method precision, specificity, sensitivity, limit of detection/quantitation, and analyte stability were evaluated. The validated methods showed excellent linearity, with a correlation coefficient > 0.99, and good precision, with intra-day precision < 7.4% for all tested analytes. The percent recoveries ranged 83–104% within the method’s quantitation range. In comparison to previously reported methods, the current method has a much shorter equilibration time, higher sensitivity, better separation for many solvents, and a wide concentration detection range. The current method is also perfectly suitable to analyze short chain fatty acids such as formic acid, acetic acid, butyric acid, and valeric acid without requiring additional extraction or derivatization steps. Notably, the method was found to be suitable for analysis of formic acid—a common solvent in certain nanoformulations and one in which there is no prior gas chromatography method available which does not require this additional sample manipulation—down to approximately 75 ppm. Herein, the method is demonstrated using various nanoformulations, including the commercial Doxil formulation as well as several research nanoformulations, including polymeric, cross-linked polymeric, and dendrimer platforms.
Synthetic mRNA produced by in vitro transcription is the active pharmaceutical ingredient of approved vaccines and of many drugs under development. It typically contains a 3' poly(A) tail required for optimal stabilization and translation of the mRNA. The average length of the poly(A) tail in the produced mRNA cannot be precisely predicted and consequently must be measured using complicated technologies such as reverse transcription and sequencing or cleavage of the poly(A) tail and analysis by capillary electrophoresis or mass spectrometry. We report an accelerated method to evaluate the average poly(A) tail length in synthetic mRNA, which benefits from the fact that thiazole orange is fluorescent only when in close proximity of nucleic acid. An oligo(dT)12 oligonucleotide having thiazole orange at its extremities emits a fluorescence signal proportional to the amount of poly(A) sequence available. Using a titration curve made with known amounts of poly(A) RNA oligonucleotide, the thiazole orange oligo(dT)12 oligonucleotide can instantly indicate the average length of the poly(A) tail in an mRNA sample. This fast and easy method can be used in any laboratory to determine the size of the poly(A) tail in in vitro-transcribed mRNA produced for research, pre-clinical studies and clinical trials.
Background: Walking is a dynamic activity that relies on inputs from the multisensory system, i.e., somatosensory, vision, and vestibular. These inputs are processed and integrated in the central nervous system to produce motor impulses for efficient walking balance. The Sensory Organization Test (SOT) is established as the gold standard for assessing sensory contributions to standing balance. However, no comparable assessments have been developed for the clinical evaluation of balance during gait. This study evaluated the Gait Sensory Interaction Test (GaitSIT), a novel virtual reality (VR)-based assessment for characterizing sensory-condition-specific changes in walking balance. Methods: The GaitSIT comprises a VR environment with a physical compliant foam walking surface that evaluates gait–balance by systematically manipulating and evaluating the sensory systems. Twenty-nine healthy young adults (mean age 24.9 ± 6.4 years) were instructed to complete 6 m walking trials under six standardized conditions (C): eyes open, eyes closed/dark scene, and rotating visual scenes on a firm surface, then repeated on a foam surface. Wearing an Oculus VR headset, participants were instructed to walk in a straight line at their preferred speed, as naturally as possible, in two test sessions on the same day, followed by a third test session 24 h later. Headset-derived sway measures, including position, velocity, and acceleration data, were recorded, and the continuous trajectory deviation angle (i.e., directional control) and sensory ratios were calculated. Linear mixed-effects models included trial-level walking speed as a covariate. Additionally, participants completed the modified Clinical Test of Sensory Interaction on Balance (mCTSIB) as a clinical standing-balance reference measure; its concurrent-validity findings will be reported separately. Results: Significant condition effects were observed for position, velocity, acceleration, and CTDA after adjustment for trial-level walking speed (all p<0.001), indicating that the six sensory conditions elicited distinct gait–balance responses. Significant differences relative to the baseline condition (C1) were observed across conditions C2–C6 for position, C3–C6 for velocity, and C2 and C5 for acceleration. Session effects were not significant for any primary kinematic outcome after speed adjustment. A significant condition × session interaction was observed for position (p<0.001), whereas velocity, acceleration, and CTDA demonstrated no significant interactions. Walking speed was significantly associated with position, acceleration, and CTDA, but not velocity. Sensory-ratio analyses revealed larger visual and vestibular ratios relative to somatosensory ratios, with the visual and vestibular ratios generally decreasing across sessions. Conclusions: GaitSIT successfully manipulated sensory conditions during overground walking and produced significant changes in gait-related sway, directional control, and sensory-ratio measures. These findings support the feasibility of GaitSIT as a portable, low-cost, and immersive assessment framework for characterizing sensory-condition-specific gait–balance responses after accounting for walking speed and providing indirect behavioral indices related to sensory reweighting.
Background: Carbon dots (CDs) are promising fluorescent nanomaterials with great application potential in bioimaging and organelle-targeted diagnostics. This study compares nitrogen-doped (N-CDs) and boron–nitrogen co-doped CDs (BN-CDs) in normal NIH3T3 fibroblasts and KRAS-transformed cells. Methods: CDs were synthesized via a microwave-assisted method. Their fluorescence, cytocompatibility, and intracellular localization were evaluated using confocal microscopy, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assays, and organelle colocalization. Cellular metabolism was assessed by Seahorse analysis. Oxidative stress and cAMP levels were pharmacologically modulated. Results: BN-CDs exhibited stronger intracellular fluorescence than N-CDs, indicating enhanced uptake and imaging performance, with no cytotoxicity up to 100 µg/mL. They localized to multiple organelles, particularly mitochondria. However, fluorescence was significantly reduced in KRAS-transformed cells despite similar mitochondrial mass. BN-CDs did not affect mitochondrial respiration or glycolytic activity. Induced oxidative stress or elevated cAMP in normal cells reduced BN-CD fluorescence. Conclusions: Boron doping improves N-CD imaging properties without affecting cell viability or metabolism. Reduced fluorescence in KRAS cells is associated with altered intracellular conditions, suggesting that BN-CDs could be used to discriminate between normal and cancer cells.
Potato (Solanum tuberosum L.) is an important staple and food security crop to many communities in the world. However, potato production and quality is greatly constrained by bacterial wilt, a disease caused by a soil-borne pathogen, Ralstonia solanacearum. Ralstonia solanacearum can be managed through clean seed systems and therefore laboratory testing is a pre-requisite for seed certification to confirm the absence of the pathogen in potato seeds before planting. Molecular diagnostics is the gold standard for detection of R. solanacearum in potato seeds. However, the extraction of genomic DNA from R. solanacearum for molecular diagnostics is complex, tedious, lengthy and/or costly procedure. A simple, rapid and reliable DNA extraction protocol is required for use in routine molecular diagnosis of R. solanacearum, a high-risk quarantine pathogen. In this study, we developed a simple and rapid protocol for extracting genomic DNA from symptomatic and asymptomatic potato tubers infected with R. solanacearum and verified its efficiency for the detection and molecular characterization of the pathogen. The protocol was developed from the evaluation of distilled water, Tris-EDTA (TE) and Tris buffer as a base solution for tissue maceration. The DNA quantity and integrity was determined using the NanoDrop 2000C spectrophotometer and agarose gel electrophoresis, respectively. Both hot and cold solutions produced intact high molecular weight genomic DNA of sufficient yield and purity for molecular-based applications. The detection and determination of phylotypes of R. solanacearum, based on conventional and multiplex polymerase chain reaction (PCR), amplified the expected 280 and 372 bp amplicons, respectively, confirming that the quantity and quality of the extracted pathogen genomic DNA was sufficient for molecular diagnostic applications. The sequencing of the amplified products of the endoglucanase gene produced good quality sequences, which confirmed the R. solanacearum isolates to be members of phylotype II sequevar 1. This protocol is a simple, fast and reliable tool for the extraction of sufficient genomic DNA with high quality, directly from R. solancearum-infected potato tubers for PCR and sequencing applications. Its simplicity and throughput make it valuable for use in routine diagnostics and can be adopted by certification programs to ensure distribution of clean potato seeds to farmers.
Accurate deconvolution of bulk transcriptomes is essential for characterizing the breast cancer tumor microenvironment (TME), yet existing reference matrices incompletely capture tumor-specific cellular diversity. Here, we developed breast cancer-specific multiresolution CIBERSORTx signature matrices from single-cell RNA sequencing data and systematically evaluated their analytical performance and interpretability. Major-, minor-, and subset-level matrices were constructed and assessed using pseudo-bulk mixtures and pure cell profiles, while biological and clinical coherence were evaluated in TCGA-BRCA and the I-SPY2 cohort. All matrices demonstrated high accuracy in reconstructing pseudo-bulk compositions, with performance declining at finer resolution. Spillover increased with granularity but was largely restricted within related lineages. Lineage-wise deconvolution modestly reduced spillover but consistently decreased accuracy, highlighting the importance of cross-lineage transcriptional contrast. In external datasets, most inferred cell populations showed biologically coherent associations with canonical markers and pathways, whereas some fine-resolution subsets exhibited non-canonical patterns, likely reflecting intra-lineage trade-offs or context-dependent transcriptional states. In the I-SPY2 cohort, plasmablasts and selected myeloid populations were positively associated with pathological complete response, whereas fibroblastic and perivascular-like populations showed negative associations. These findings establish a validated and interpretable resource for breast cancer TME deconvolution and clarify its performance characteristics and limitations.
Reproducible experimental models of localized dermal-hypodermal fibrosis are essential for standardized investigation of regenerative interventions. Variability in bleomycin dosing, anatomical targeting, and assessment strategies limits cross-study comparability. This study describes a methodological framework for standardized induction of early dermal-hypodermal remodeling in a rat model followed by autologous subcutaneous tissue grafting and multimodal longitudinal evaluation. Female Wistar rats underwent subcutaneous bleomycin administration at 1 mg/kg/day for three consecutive days. Clinical documentation, high-frequency ultrasonography with fixed imaging parameters, and sequential biopsies from a predefined thoracic anatomical site were performed at baseline, intermediate reassessment, and final evaluation. Autologous subcutaneous tissue grafting was conducted at Day 17 after study initiation. The protocol enabled controlled induction of early structural remodeling and consistent longitudinal documentation of dermal-hypodermal thickness, echogenicity changes, and histological architecture within a standardized anatomical region. This protocol development study establishes a reproducible and spatially consistent experimental platform integrating imaging and histological assessment, facilitating future hypothesis-driven investigations of fibrotic remodeling and regenerative strategies.
