Poly(ADP-ribose) (PAR) is a polymer of ADP-ribose synthesized by four members of the ADP-ribose polymerase family of enzymes-PARP1, PARP2, PARP5a, and PARP5b. However, only PARP1 and PARP2 synthesize PAR in response to DNA breaks. PAR is defined as a protein post-translational modification, but it is also shown to exist as a DNA or RNA modification. Levels of PAR are further regulated by PARG, a PAR glycohydrolase that, together with PARP1 and PARP2, modulates the cellular level of DNA damage-induced PAR. The dynamic synthesis and degradation of PAR is critical to its regulatory role in DNA repair, and the DNA damage response, which in turn affects chromatin reorganization, replication, transcription, and cell death. PARP1/PARP2 activation and the accumulation of PAR can be considered sites of ongoing base excision repair or DNA single-strand break repair; however, numerous PARP1/PARP2 activators are also associated with replication stress and other DNA metabolic processes. Once formed, PAR chains facilitate the recruitment of DNA repair and DNA damage response (DDR) factors to sites of DNA damage or genomic insult via their PAR-binding domains (PBDs). Ten different PBDs recognize various regions of the PAR molecule, including the PAR binding motif, PAR binding Zinc finger, the WWE domain, and the macrodomain, among other PBDs. To facilitate cellular analysis of PAR dynamics, we used PBDs fused to enhanced green fluorescent protein (EGFP) to optimize cell-based quantitation of PAR foci. We describe an assay that uses a fragment of RNF146 encoding the PBD/WWE domain, linked to EGFP, to visualize and quantify PAR accumulation at sites of genomic insult and ongoing BER or SSBR. We describe experimental steps, including the production of lentiviral particles, transduction of the target cell line, treatment of mammalian cells to induce genomic DNA damage, acquisition of confocal fluorescence micrographs, and semi-automated quantification of the data.
The prognosis of lung adenocarcinoma (LUAD) is traditionally evaluated via tumor-node-metastasis (TNM) staging, which does not account for the patient's immune status. This protocol integrates genomic profiling and immune microenvironment analysis to facilitate a more comprehensive postoperative prognostic evaluation. The method involves a retrospective analysis of paraffin-embedded tumor tissues using two primary techniques. First, next-generation sequencing (NGS) is performed with a customized 37-gene panel to identify mutations in driver genes and variants of uncertain significance. Second, multiplex immunofluorescence (mIF) is utilized to target markers including HLA-DR, CD68, CD163, CD206, PD-L1, and PanCK. This enables the quantification of spatial distribution and density for specific immune cell subpopulations across various tumor regions. This integrated approach enables the simultaneous assessment of genomic heterogeneity and tumor-infiltrating immune cell characteristics. The resulting data identifies specific combinations of mutational profiles-such as EGFR status-and immune cell densities. These integrated combinations enable the study of their collective impact on patient survival, offering a promising approach for the development of future lung cancer prognostic models. This protocol demonstrates a robust method for characterizing the complex biological features of the LUAD tumor microenvironment.
Oxygen-sensitive optoacoustic imaging (OS OAI) and dynamic contrast-enhanced optoacoustic imaging (DCE OAI) provide complementary, noninvasive readouts of tissue physiology. OS OAI leverages multispectral detection of endogenous oxyhemoglobin and deoxyhemoglobin to generate maps of blood oxygen saturation (%sO₂), while DCE OAI tracks the in vivo pharmacokinetics of a near-infrared absorber to quantify vascular perfusion and permeability. The goal of this protocol is to present a unified OS-DCE OAI workflow that enables simultaneous assessment of oxygenation and perfusion in two widely used pre-clinical settings: orthotopic breast cancer tumors and full-thickness cutaneous wounds. In this protocol, we demonstrated OAI of mouse models of breast cancer or a laceration wound. For OS OAI, multi-slice multispectral scans are acquired at 700-875 nm to allow spectral unmixing and estimation of %sO₂. For DCE OAI, repeated acquisitions at ≤5-s temporal resolution are performed before and after intravenous bolus injection of indocyanine green (ICG), using either multispectral sampling at 700-875 nm or single-wavelength sampling at 800 nm to generate time versus OA signal amplitude curves. The development of animal models, preparation for OA imaging, OS OAI and DCE OAI acquisitions, and analyses to measure characteristics of oxygenation and vascular perfusion are detailed, along with key troubleshooting guidance for motion artifacts, superficial absorbers, and failed injections. Together, this OS-DCE OAI protocol yields an integrated functional portrait of hypoxia and vascular transport that can detect physiological changes in tumors and wounds earlier than gross anatomical measures. OS-DCE OAI is readily adaptable to other disease models.
This study assessed the risk of active pulmonary tuberculosis (ATB) in elderly patients with weakened immunity by jointly detecting serum interferon-γ (IFN-γ) and neutrophil CD64, and analyzed the impact of this diagnostic protocol on the course of ATB. A total of 50 patients with ATB and 50 age- and sex-matched healthy controls were recruited. Subsequently, the patient's neutrophil CD64 (quantified as the percentage of CD64-positive neutrophils [CD64%]), IFN-γ (by enzyme-linked immunosorbent assay [ELISA]), C-reactive protein (CRP; by immunoturbidimetry), and erythrocyte sedimentation rate (ESR; by Westergren method). Sputum samples from patients were cultured on Löwenstein-Jensen (L-J) medium and in the MGIT 960 automated system. The diagnostic efficacy of individual and combined biomarkers was assessed with receiver operating characteristic (ROC) curve analysis, and a logistic regression model was developed for combined detection. Significantly elevated levels of both IFN-γ and CD64 were observed in ATB patients versus healthy controls (P<0.05). A diagnostic model incorporating both biomarkers had an area under the curve (AUC) of 0.838, with a sensitivity of 84.00% and a specificity of 76.00%. Both markers decreased following therapeutic intervention (P<0.05), showing the lowest values in culture-negative patients (P<0.05). Furthermore, the combined model showed predictive utility for culture conversion, attaining an AUC of 0.756 (65.71% sensitivity, 80.00% specificity; P<0.001). The combined detection of IFN-γ and CD64 can effectively diagnose the occurrence of ATB in immunocompromised elderly people, providing a reference for clinical practice.
The contractility of cLVs is the driving force for lymph flow and a key mechanism underlying brain toxin cleansing. Here, we present an innovative method for in vivo two-photon imaging of the contractility of cLVs and the long-term observation of the removal of red blood cells with lymph flow in cLVs in mice. The components of the setup include a mini-heating pad adapted to the mouse's neck that allows for increasing the window for imaging up to 5 h while preserving the cLV contractility and the lymph flow; humidity control; a protective casing; and a positioning system that allows for maintaining stable optical visualization of cLVs. The method ensures reproducibility and compatibility with other types of two-photon microscopes and a wide range of objectives, maintaining high-quality images with depth up to 500 µm. The protocol includes detailed stages of surgical preparation of cLVs for maintaining the physiological environment, providing the normal cLV contractility.
The performance of lead-acid batteries is highly dependent on the structural and surface properties of the electrodes' active materials. Besides the importance of the material's phase composition at any given time in application, its surface area and porosity contribute to the overall performance of the battery. Mercury porosimetry, which uses a representative sample of the active material for its analysis, has traditionally been used to measure the porosity of battery plates. Apart from the possibility of inducing cracks and cavities in the material while taking the sample, many laboratories have moved away from the use of mercury due to its toxicity. The Archimedes-based glycerol displacement technique has the advantage of being a simple method that considers the porosity of the entire battery plate. Even though the method might not give detailed information on the material's pore volume distribution, reproducibility is achieved for individual plates as well as groups of plates taken from either a single cell or six cells from a battery. The method calculates porosity based on the material's absolute and envelope density, which makes use of absorbed glycerol volume to measure the average porosity over the entire plate. Lead-acid batteries are usually subjected to aging mechanisms that affect the active material's structural integrity and surface properties. This study describes the use of the glycerol displacement technique for the measurement of battery plate porosities, combined with powder X-ray diffraction material characterization to understand the degradation mechanisms of batteries that were subjected to capacity life cycling and calendar aging or shelf life, respectively. The results showed that the failure mechanisms of batteries are significantly different and that both the chemical and physical properties of the active material are interdependent; therefore, one cannot rely on a single analytical tool only to interpret a failure mechanism.
This paper presents a computational bench-marking assessment of Ensemble Learning algorithms in the prediction of heart disease, combining different Machine Learning algorithms, such as hard voting, soft voting, and stacking, in a single framework. The evaluation was conducted using publicly available cardiovascular dataset obtained from the Kaggle repository (https://www.kaggle.com/datasets/sid321axn/heart-statlog-cleveland-hungary-final) comprising 1,190 instances and 11 clinical features. The process involves data preprocessing, which includes handling missing values, removing outliers, scaling variables and class balancing to ensure uniform input feature selection, based on Random Forest (RF), is used to eliminate unnecessary features. Among the evaluated models, the stacking ensemble classifier achieved the highest overall accuracy of 91.88% on the test dataset. Although additional metrics such as precision, recall and F1-score were computed for comparative analysis, the emphasis of this study remains on methodological benchmarking rather than clinical validation. Various base classifiers, including Decision Tree, Random Forest, AdaBoost, and XGBoost, are applied and tested independently. These models are then combined using ensemble techniques with hard voting, soft voting, and stacking. In stacking, Logistic Regression is used as the meta-model, which is trained on cross-validated predictions of the out-of-fold samples to avoid overfitting. Evaluations are carried out using accuracy as the primary criterion for comparison, so that individual classification systems and their combination strategies can be compared uniformly in the same preprocessing and validation environment. Though performance metrics are provided for comparative indications, the emphasis of the approach lies in the development and evaluation of strategies and not in their clinical assessment. This protocol makes it easy to compare ensemble machine learning algorithms on publicly available cardiovascular datasets and helps to make a systematic comparison of data preprocessing and ensemble configuration approaches.
Lumbar disc herniation (LDH) is commonly caused by annular disruption resulting from trauma or poor posture, leading to extrusion of nucleus pulposus material and compression of lumbar nerve roots. This condition often presents with radicular pain and sensory disturbances radiating from the lower back to the lower extremities. Although most cases can be managed conservatively, severe or refractory symptoms may require surgical intervention, including conventional discectomy, minimally invasive microscopic discectomy, and endoscopic discectomy. Percutaneous endoscopic lumbar discectomy (PELD) can be performed under local anesthesia; however, the procedure relies heavily on the surgeon's experience and is associated with technical challenges, including positioning difficulty, repeated fluoroscopic confirmation, and increased radiation exposure. The objective of this study was to develop and evaluate a novel percutaneous endoscopic lumbar disc positioning device designed to improve the precision and efficiency of disc positioning during PELD. This study developed a unique percutaneous endoscopic lumbar disc positioning device. The device was realized through the reconstruction and repair of 3D models of the porcine spine and lumbar spine, fabrication of a convex base plate, design of spinal spinous process and disc positioning guiding devices, computer-aided design of a percutaneous endoscopic lumbar disc minimally invasive surgical navigation module, and accuracy testing. Experimental results showed that the device could accurately locate positions on porcine spines and may reduce fluoroscopic dependence in experimental settings. The positioning device provided high precision, supporting minimally invasive surgery by reducing incision size, avoiding damage to surrounding critical blood vessels and nerve tissues, and offering multi-directional surgical instrument guiding paths, facilitating the surgical process. The percutaneous endoscopic lumbar disc positioning device described in this study provides a structured, reproducible approach to intervertebral disc positioning during PELD. This method has the potential to improve procedural efficiency, limit radiation exposure, and support surgical training, particularly for early-career spine surgeons.
Three-dimensional (3D) co-culture is a rapidly evolving technique for researchers looking to accurately study cell-cell interactions using in vitro experiments. The limitations of monolayer cell culture, including limited interactions between the cellular and extracellular environment and disturbed cell morphology, are addressed by simulating the in vivo cellular environment. Using a scaffold to provide structural support and including biologically active extracellular matrix components, 3D cultures display behaviours and morphologies more congruent with tissue. Incorporating multiple cell types into this kind of 3D environment allows for the study of cell-cell interactions inside a biomimetic model system. A wide range of 3D co-culture technologies has emerged, each with its own advantages and challenges. Often these technologies require specialized equipment, a complex setup, or specific technical knowledge. As well, there exist a few standardized methods for studying indirect cell-cell interactions between two cell types separated by a reconstituted basement membrane. Here, we describe a 3D co-culture method that requires only fundamental technical skills and uses more widely applicable materials to successfully recapitulate indirect cell-cell interactions across a basement membrane. A monolayer of cells is covered in a layer of extracellular matrix, in the form of Matrigel, and a second cell type is seeded on top. The resultant co-culture is maintained for five days, at which point cells are analyzed for morphological changes by immunofluorescence or extracted from the co-culture for more detailed genomic, transcriptomic, or proteomic analyses. This protocol is ideal for studying the impact of cell-cell communication on cell behaviour when physical contact is prohibited by a basement membrane. As researchers continue to opt for more in vivo-relevant cell culture methods, a streamlined approach is necessary to avoid high barriers to entry.
Vascularized composite allotransplantation (VCA) is increasingly recognized as a reconstructive option that offers substantial benefits for patients with severe tissue loss, including injuries affecting the face and hand. Despite its clinical promise, VCA remains limited by surgical complexity, the need for long-term immunosuppression, and associated morbidity. Preclinical models are therefore essential for refining surgical techniques, testing novel immunomodulatory approaches, and investigating ischemia/reperfusion injury. Compared with large-animal models, rodent models offer advantages in accessibility and cost while adhering more closely to the 3Rs principles of Replacement, Reduction, and Refinement. Among them, the mouse offers unique advantages, including the availability of humanized strains, a broad panel of well-characterized antibodies, and compatibility with advanced immunological assays. These features make the mouse particularly valuable for translational VCA research. Murine VCA, however, requires demanding supermicrosurgical skills, as vascular anastomoses are performed on vessels measuring approximately 0.3-0.5 mm in diameter. To enhance reproducibility, we provide technical tips that have proven critical to the success of these procedures. These include the insertion of a nylon filament into the vessel lumen to stabilize the anastomosis, careful separation of the artery and vein over a sufficient distance along the pedicle, precise anesthetic dosing, and specific maneuvers to prevent pedicle twisting. We also highlight common pitfalls and errors, offering practical guidance to improve outcomes. Here, we describe a comprehensive, stepwise protocol for mouse heterotopic hindlimb-to-neck transplantation, accompanied by instructional video material. This non-functional model minimizes postoperative morbidity compared with orthotopic transplantation while providing robust and reproducible results. It is ideally suited for training in supermicrosurgery and for addressing key experimental questions in VCA, including immune tolerance, graft preservation, and ischemia/reperfusion mechanisms. This methodology provides investigators with a reliable murine platform to advance translational VCA research and to develop innovative strategies aimed at improving outcomes in reconstructive transplantation.
High-risk corneal transplantation in patients with glaucoma, keratoconus, or prior graft failures is frequently associated with suboptimal outcomes. Although individual determinants such as age, gender, diabetes, and intraocular pressure (IOP) are recognized risk factors, their collective impact on graft survival has not been systematically quantified. This meta-analysis aims to clarify the role of glaucoma, keratoconus, and key clinical factors in shaping the prognosis of high-risk corneal transplantation. A systematic search of PubMed, Cochrane, Embase, and Web of Science databases was conducted to identify studies on high-risk corneal transplantation involving glaucoma and keratoconus. Data from 11 observational studies (10,558 cases) were analyzed. Across the included studies, transplant success was generally defined as graft survival with preserved corneal clarity. Subgroup analyses were performed to evaluate the impact of demographic and clinical determinants on transplant success. Glaucoma was found to significantly impact transplant success, with an odds ratio (OR) of 1.43 (95% CI [1.26, 1.63], p < 0.001), while keratoconus also represented a risk factor (OR = 1.22, 95% CI [1.13, 1.31], p < 0.001). Subgroup analysis for glaucoma patients indicated that younger age (<60 years), female gender, absence of diabetes, and preoperative IOP ≤25 mmHg were favorable factors for transplant success. In keratoconus patients, those under 30 years old and female had better transplant outcomes. Sensitivity analysis confirmed the robustness of these results, and minimal publication bias was observed. This study demonstrates that advanced age, male gender, diabetes, and elevated preoperative IOP significantly compromise the success of high-risk corneal transplantation. These findings provide robust evidence to guide patient selection, preoperative optimization, and surgical decision-making in high-risk populations. Incorporating these determinants into clinical practice may enhance graft survival and visual outcomes, while informing the development of tailored management strategies and future clinical guidelines.
This protocol details an optimized method for the production of small stable spheroids, their culture, and 3D imaging, for the study of the endothelial and insulin-producing β cells interactions in a 3D model of pancreatic islets. The 150-200 µm spheroids, mirroring the lowest range of islet sizes, were prepared from a selected ratio combining 1 intra-islet endothelial cells (MS-1 cells) to 20 insulin-secreting cells (β-TC-6). Staining, clearing, and mounting challenges of small spheroids and their tackling by employing low-melting point agarose and the CUBIC clearing technique are detailed, as well as key points for an efficient analysis of the 3D structure with different probes. Data indicate that NTPDASE-ectonucleotidase 3 does not colocalize with insulin in the spheroid model, suggesting varying maturity and functional levels of β-TC6 and that the complete procedure can also be applied to isolated pancreatic islets, with clear probing of intra-islet vessels. These findings underscore the effectiveness of the 3D imaging protocol in revealing complex pancreatic cell organization and interactions within the islet model.
This work proposes a reproducible computational protocol for multimodal affective modeling that utilizes physiological signals. The goal of the protocol is to enable offline emotion recognition by integrating multiple bio signals using a unified deep learning framework. The proposed work consists of five steps: data collection, preprocessing, feature alignment, multimodal fusion, and evaluation. EEG, ECG, and GSR signals from publicly accessible AMIGOS data were used as the experimental baseline in this work. Bio signals were pre-processed and normalized to extract modality-specific features. Heterogeneous feature spaces were aligned across modalities using Deep Canonical Correlation Analysis, followed by a multimodal fusion network for classifying an affective state. The protocol has been evaluated with offline experiments and compared to conventional fusion and classification models using standard performance metrics such as accuracy, precision, recall, F1-score, and AUC. This study focuses on the development and validation of a computational framework for multimodal affective user experience modeling rather than the deployment of a real-time interactive system. With 92.1% accuracy for UX-affective state prediction and 94.2% F1-score for valence-arousal classification, the results consistently outperformed baseline models on emotional dimensions. These findings verified the effectiveness of the proposed multimodal fusion workflow for computational affective modeling by benchmarking physiological data.
This method enables the identification of test substances that inhibit DIO1 activity and may thus interfere with thyroid hormone homeostasis. The protocol presented describes a non-radioactive, colorimetric method for assessing the effects of test substances on the enzymatic activity of Deiodinase 1 (DIO1) in human liver microsomes. The assay is based on the Sandell-Kolthoff (SK) reaction, which quantifies iodide released during the deiodination of reverse triiodothyronine (rT3) to diiodothyronine (T2). The yellow cerium (IV) is reduced to colorless cerium (III) in the presence of iodide, allowing photometric detection at 415 nm. In this standardized setup, the cerium (IV) concentration is set to 40 mM to improve signal stability and robustness under the described conditions. To ensure assay reliability and reproducibility, a comprehensive control setup is included: 6-propyl-2-thiouracil (reference item), aurothioglucose (positive control), 1-thio-β-D-glucose sodium salt (negative control), and 1% dimethyl sulfoxide (solvent control). The protocol includes a range-finding assay to determine appropriate test concentrations, standardization of microsome batch-specific iodide release activity and structured chemical interference testing in the absence of microsomes, with an optional follow-up assessment without dithiothreitol (DTT) to identify DTT-dependent artifacts. The DIO1-SK assay is suitable for medium- to high-throughput screening and mechanistic toxicology studies. Its robustness, scalability, and applicability to human microsomes make it a valuable tool for investigating endocrine disruption. This protocol supports regulatory validation efforts.
Research on endometrial disorders, including tamoxifen-associated endometrial hyperplasia, has been limited by the lack of robust in vitro epithelial models. Conventional methods for isolating human endometrial epithelial cells (HEECs) are often inefficient, technically demanding, and poorly scalable. Here, a simplified, feeder-free protocol is described that integrates mixed-enzyme digestion, erythrocyte lysis, and sustained ROCK inhibitor (Y-27632) treatment to enable efficient isolation and expansion of primary HEECs. The inclusion of Y-27632 is critical for mitigating dissociation-induced apoptosis and enhancing early-stage cell adhesion. This approach supports serial passaging while maintaining epithelial identity for at least 3 passages, with morphology preserved beyond 10 passages. Characterization by Western blot and immunofluorescence confirms high expression of Cytokeratin 18 and E-cadherin, with a progressive reduction in vimentin-positive stromal contaminants under selective culture conditions. By addressing key technical limitations, this method provides a reliable, scalable source of epithelial cells for mechanistic studies, disease modeling, drug screening, and regenerative applications in endometrial research. The standardized workflow offers an alternative to complex co-culture systems, facilitating broader access to high-quality primary cell models.
Laser capture microdissection (LCM) provides spatial access to specific cell populations within complex tissues through in situ visualization and isolation. To enable transcriptomic analysis of histologically defined regions in fixed tissue, a detailed LCM protocol is presented for RNA extraction from paraformaldehyde (PFA)-fixed, Optimal Cutting Temperature (OCT) compound-embedded mouse liver sections. The protocol details the identification and collection of microscale samples (approximately 1,000 cells) through a workflow encompassing tissue fixation, sucrose dehydration, OCT embedding, cryosectioning, hematoxylin staining, and laser-capture of targeted histological areas. Using this method, a high-purity RNA (A260/A280: 1.9-2.1) was obtained. The RNA integrity number (RIN) was 6.7 ± 0.9, reflecting the expected fragmentation associated with PFA fixation. However, quantitative PCR for β-actin yielded Ct values of 17-19, and RNA sequencing performed using fragmentation-optimized library preparation generated high-quality reads, with >90% of bases meeting Q20 and Q30 thresholds, confirming that the RNA is suitable for sensitive downstream analyses. Therefore, this protocol enables spatially resolved, targeted gene expression analysis by providing RNA of defined purity and integrity from specific histological regions of PFA-fixed liver tissue.
The study aims to delineate the evolutionary trajectory, global collaborative networks, intellectual structure, and shifting clinical frontiers of the induced membrane technique (IMT, Masquelet) for managing osteomyelitis and severe infected bone defects. Publications from 2001 to 2026 were retrieved from the Web of Science Core Collection database. Bibliometric and visualization analyses were conducted using R Bibliometrix. A carefully curated dataset of 307 foundational original articles from 40 countries was included. The logistic growth model (R2=0.827) indicates the field is transitioning from a period of novel expansion into clinical maturation. Geographically, France pioneered the foundational high-impact literature, whereas China has recently experienced an exponential surge in publication volume, albeit operating largely within domestic research silos with limited international crosstalk. Author co-citation algorithms unmasked three distinct theoretical pillars driving the field: the foundational anatomical and physiological conceptualization of the induced membrane, the ongoing clinical dialectic against distraction osteogenesis (the Ilizarov bone transport paradigm), and the rigorous integration of classical Cierny-Mader osteomyelitis principles. Crucially, thematic mapping and Sankey evolution revealed a profound clinical paradigm shift. Early historical enthusiasm for biological modifiers has been aggressively replaced by contemporary pragmatic limb salvage strategies (2022-2026), heavily characterized by burst keywords such as flap and amputation. Bibliometric analysis reveals that IMT research has evolved from early technique validation into a core strategy for complex multidisciplinary extremity salvage. Current frontiers emphasize aggressive infection eradication and massive composite defect management. Future advancements necessitate transnational multicenter trials to establish standardized clinical protocols.
Pulmonary rehabilitation (PR) is a core component of chronic obstructive pulmonary disease (COPD) management; however, its implementation during acute exacerbations remains variable, particularly with respect to individualized approaches. This study aimed to evaluate the association between inpatient personalized PR and recovery-related clinical outcomes in hospitalized patients with acute exacerbations of COPD (AECOPD). A single-center retrospective cohort study was conducted at a tertiary care hospital between January 2021 and June 2024. Hospitalized patients aged ≥40 years with spirometry-confirmed COPD who experienced AECOPD and completed an inpatient personalized PR program were included. The rehabilitation program was initiated within 48-72 h of clinical stabilization and tailored based on baseline symptom burden, functional capacity, dyspnea severity, oxygen saturation, and exercise tolerance. Primary outcomes included changes in the COPD assessment test (CAT), modified medical research council (mMRC) dyspnea scale, and 6-minute walk test (6MWT) distance from baseline to program completion. Secondary outcomes included length of hospital stay and 30-day readmission rates. A total of 200 patients were included in the analysis. Mean CAT scores decreased from 25.4 ± 4.6 at baseline to 17.2 ± 3.8 following rehabilitation (p < 0.001). Mean mMRC dyspnea scores improved from 3.1 ± 0.8 to 2.0 ± 0.7 (p < 0.001). Functional exercise capacity increased, with the mean 6MWT distance improving from 210 ± 68 m to 310 ± 75 m (p < 0.001). The average length of hospital stay was reduced from 10.5 ± 3.2 days to 6.3 ± 2.1 days (p < 0.001), and 30-day readmission rates decreased from 25% to 10% (p < 0.001). In this retrospective cohort, inpatient personalized PR implemented during AECOPD was associated with improvements in symptom burden, dyspnea severity, functional exercise capacity, and selected healthcare utilization outcomes.
This case report demonstrates the removal of an intravesical Gellhorn pessary and management of a large vesicovaginal fistula. A 79-year-old woman with a history of pelvic organ prolapse treated with a Gellhorn pessary three years prior presented with recurrent urinary tract infections and continuous urinary leakage. CT imaging showed the pessary to be intravesical. After surgical clearance, she underwent cystoscopy and vaginoscopy under anesthesia with the goal of transvaginal removal. The pessary was fully embedded in the bladder lumen and could not be mobilized using laser transection, cutting, or grasping techniques. A guidewire was passed through a pessary drainage port and retrieved endoscopically, allowing counter-traction to gradually mobilize the pessary sideways. It was grasped with clamps and removed transvaginally through the large fistula site. Two-month follow-up cystoscopy showed resolution of bladder inflammation. MRI with vaginal contrast demonstrated a persistent large vesicovaginal fistula. Due to age and medical frailty, she underwent Latzko colpocleisis with suprapubic catheter placement for bladder drainage. At four weeks, cystography confirmed fistula closure. At the last follow-up, the patient reported no leakage, normal voiding, and resolution of infections.
This protocol describes a sequential differential centrifugation approach to enrich lipid droplets (LDs) of different sizes from bovine mammary epithelial cells (BMECs) and mammary tissues, enabling size-resolved analysis beyond conventional bulk LD isolation methods. As dynamic regulatory hubs of intracellular lipid metabolism, LDs serve critical functions by storing neutral lipids (such as triglycerides and cholesteryl esters) and coordinating their synthesis, hydrolysis, and transport. They play a central role in maintaining energy homeostasis, supporting membrane biogenesis, and facilitating cellular signal transduction. LD extraction is essential for investigating the regulatory mechanisms of lipid metabolism. Studying mammary gland lipid metabolism has significant implications for infant development, human health, agricultural economics, and fundamental cell biology. Therefore, we describe a differential centrifugation method for extracting LDs from BMECs and mammary gland tissue. In contrast to conventional bulk LD isolation approaches, this protocol enables size-based enrichment of LD subpopulations by sequentially adjusting centrifugal forces. The integrity and relative enrichment of LD fractions are preliminarily evaluated using BODIPY493/503 staining, providing a practical and reproducible approach for downstream analyses of lipid metabolism and LD-associated processes.