Search Results (275)

Nucleic acid aptamers leverage defined tertiary structures for precise molecular recognition, positioning them as transformative biomedical tools. We engineered AP1-F, a G-quadruplex (G4)-structured aptamer that selectively binds membrane-anchored nucleolin (NCL) non-permeabilizing, overcoming a key limitation of conventional probes. Microscale thermophoresis confirmed nanomolar affinity to NCL. By means of rigorous optimization, AP1-F attained a greater than ten-fold fluorescence signal ratio between malignant and normal cells in co-cultures, exceeding the extensively researched AS1411. Dual-channel flow cytometry demonstrated over 98.78% specificity at single-cell resolution within heterogeneous cell populations, owing to AP1-F’s unique membrane localization—unlike AS1411’s intracellular uptake, which elicited erroneous signals from cytoplasmic NCL. Competitive binding experiments and Laser Confocal Imaging confirmed that AP1-F specifically identifies cancer cells by binding to the NCL recognition site on the membrane. In pathological sections, AP1-F exhibited a 40.5-fold fluorescence intensity ratio between tumor and normal tissue, facilitating accurate tissue-level differentiation. Significantly, it delineated molecular subtypes by associating membrane NCL patterns with morphometric analysis: luminal-like MCF-7 displayed consistent staining in cohesive clusters, whereas basal-like MDA-MB-468 revealed sporadic NCL with irregular outlines—characteristics imperceptible to intracellular-targeted antibodies, thus offering subtype-specific diagnostic insights. This combination biochemical–morphological approach accomplished subtype differentiation with a single-step, non-permeabilized process that maintained lower cytotoxicity and tissue integrity. AP1-F enhances diagnostic accuracy by utilizing spatial confinement to eradicate intracellular interference, connecting molecular specificity to intraoperative margin evaluation or biopsy categorization. Full article

Mesenchymal stem cells (MSCs) are known to assist liver regeneration. In this study, we show a dose-dependent mode of recovery from liver fibrosis after intravenous injections of MSCs. Male Wistar rats experienced a 42-day-long modeling of liver fibrosis via CCl₄ poisoning and received either a single injection of 2.5 × 10⁶ MSCs on Day 3 after the last CCl₄ dose or two MSC injections on Days 3 and 10. We dynamically monitored levels of liver cytolysis markers and cytokines in the venous blood and performed a histological study of Mallory-stained liver sections. All experimental groups experienced a nearly complete recovery of biochemical markers up to 4 weeks after the end of CCl₄ administration, although we observed anti-inflammatory changes in the cytokine levels only in animals treated by two MSC injections. Histological study revealed minor signs of liver damage up to Day 90 in animals receiving two MSC doses with worse pathology in those who received a single MSC dose. Morphometric values stayed consistent with visual data, demonstrating a significantly larger number of binuclear hepatocytes, a smaller number of false lobules, and a lesser area of connective tissue proper in animals treated by two MSC injections. Our results reflect MSC grafting in applied doses to affect liver fibrosis in a dose-dependent mode. These findings provide a deeper understanding of MSC action in liver fibrosis, and the doses applied may serve as a milestone for further studies in humans. Full article

Background: Segmenting nerve fibres in histological images is a tricky job because of how much the tissue looks can change. Modern neural network architectures, including U-Net and transformers, demonstrate varying degrees of effectiveness in this area. The aim of this study is to conduct a comparative analysis of the SegFormer, VGG-UNet, and FabE-Net models in terms of segmentation quality and speed. Methods: The training sample consisted of more than 75,000 pairs of images of different tissues (original slice and corresponding mask), scaled from 1024 × 1024 to 224 × 224 pixels to optimise computations. Three neural network architectures were used: the classic VGG-UNet, FabE-Net with attention and global context perception blocks, and the SegFormer transformer model. For an objective assessment of the quality of the models, expert validation was carried out with the participation of four independent pathologists, who evaluated the quality of segmentation according to specified criteria. Quality metrics (precision, recall, F1-score, accuracy) were calculated as averages based on the assessments of all experts, which made it possible to take into account variability in interpretation and increase the reliability of the results. Results: SegFormer achieved stable stabilisation of the loss function faster than the other models—by the 20–30th epoch, compared to 45–60 epochs for VGG-UNet and FabE-Net. Despite taking longer to train per epoch, SegFormer produced the best segmentation quality, with the following metrics: precision 0.84, recall 0.99, F1-score 0.91 and accuracy 0.89. It also annotated a complete histological section in the fastest time. Visual analysis revealed that, compared to other models, which tended to produce incomplete or excessive segmentation, SegFormer more accurately and completely highlights nerve structures. Conclusions: Using attention mechanisms in SegFormer compensates for morphological variability in tissues, resulting in faster and higher-quality segmentation. Image scaling does not impair training quality while significantly accelerating computational processes. These results confirm the potential of SegFormer for practical use in digital pathology, while also highlighting the need for high-precision, immunohistochemistry-informed labelling to improve segmentation accuracy. Full article

Background: Phosphaturic mesenchymal tumours secreting fibroblast growth factor 23 (hereinafter referred to as FGF23+ PMT) are rare neoplasms that can cause hypophosphataemic osteomalacia, owing to excessive FGF23 production. Mast cells (MCs) play a key role in tumour biology by modulating proliferative activity of atypical cells, resistance to innate and acquired immunity, angiogenesis, and metastatic behaviour. However, MCs associated with FGF23+ PMT have not previously been investigated. This study, to our knowledge, is the first to characterise features of the tumour microenvironment through spatial phenotyping of the immune and stromal landscape, together with histotopographic mapping of intercellular MC interactions with other subcellular populations in FGF23+ PMT. Methods: Histochemical staining (haematoxylin and eosin, toluidine blue, Giemsa solution, picro-Mallory protocol, silver impregnation), as well as monoplex and multiplex immunohistochemical staining with spatial phenotyping, were performed to detect atypical FGF23-secreting cells, immune cells (CD3, CD4, CD8, CD14, CD20, CD38, CD68, or CD163), stromal components (CD31, α-SMA, or vimentin), and specific MC proteases (tryptase, chymase, or carboxypeptidase A3). Bioinformatics analysis using artificial intelligence technologies was applied for spatial profiling of MC interactions with tumour, immunocompetent, and stromal cells in the tumour microenvironment. Results: Bioinformatic analysis of the entire tumour histological section, comprising over 70,000 cells stained using monoplex and multiplex immunohistochemical protocols, enabled identification of more than half of the cell population. The most abundant were CD14+ (30.7%), CD163+ (23.2%), and CD31+ (17.9%) cells. Tumour-associated MCs accounted for 0.7% of the total pool of immunopositive cells and included both mucosal and connective tissue subpopulations, predominantly of the tryptase + chymase-CPA3-specific protease phenotype. This pattern reflected combined multidirectional morphogenetic processes in the patient’s FGF23+ PMT. More than 50% of MCs were colocalized with neighbouring cells of the tumour microenvironment within 20 μm, most frequently with monocytes (CD14+CD68+), M2 macrophages (CD68+CD163+), and endothelial cells (CD31+). In contrast, colocalization with atypical FGF23-secreting cells was rare, indicating minimal direct effects on tumour cell activity. Interaction with T lymphocytes, including CD8+, was also infrequent, excluding their activation and the development of antitumour effects. Mapping of MC histotopography validated the hypothesis of their inductive role in monocyte differentiation into M2 macrophages and probable polarisation of macrophages from M1 into M2, thereby contributing to slow tumour growth. MCs were further involved in extracellular matrix remodelling and participated in the formation of pro-osteogenic niches within the FGF23+ PMT microenvironment, leading to pathological osteoid development. Conclusions: This study demonstrated active MC participation in the evolution of the FGF23+ PMT microenvironment. The findings may be applied in translational medicine to develop novel algorithms for personalised therapy in patients with FGF23-secreting tumours, offering an alternative when surgical removal of the tumour is not feasible. Full article

Background: p16 immunohistochemistry (IHC) serves as a surrogate marker for high-risk human papillomavirus (hrHPV) and is widely used in gynecologic pathology. However, few studies have directly compared the staining performance and reproducibility of different p16 antibody clones in this context. Methods: We retrospectively evaluated 176 gynecologic tumor specimens including 42 whole slide sections and 134 tissue microarray cores from the cervix, endometrium, vulva, and ovary using three fully automated p16 IHC assays: E6H4 (Ventana/Roche), JC8 (Agilent/Dako), and 6H12 (Leica). Two pathologists independently reviewed each case, and concordance and interobserver agreement were analyzed. Sensitivity, specificity, and Cohen’s κ statistics were calculated, with E6H4 serving as the reference. Results: All three antibody clones demonstrated excellent staining performance with preserved tissue morphology and minimal background artifacts. Concordance for p16 positivity/negativity was 100% across all clone pairings (95% CI: 97.9–100%). Interobserver reproducibility was also perfect, with a κ coefficient of 1.00 (95% CI: 0.94–1.00). Minor non-block staining patterns did not impair interpretability. Conclusions: Our findings indicate that E6H4, JC8, and 6H12 clones yield comparable staining results when used in conjunction with standardized automated protocols. These results support the practical interchangeability of these clones in clinical and research settings, particularly when cost, availability, or risk management require substitution. Laboratories should continue to perform internal validation and utilize external quality assurance programs when implementing p16 IHC. Full article

Heparin-based delivery platforms have gained increasing attention in regenerative medicine due to their exceptional affinity for growth factors and versatility in structural and functional design. This review first introduces the molecular biosynthesis and physicochemical diversity of heparin, which underpin its binding selectivity and degradability. It then categorizes the delivery platforms into microspheres, nanofibers, and hydrogels, with detailed discussions on their fabrication techniques, biofunctional integration of heparin, and release kinetics. Special focus is given to stimuli-responsive systems—including pH-, enzyme-, redox-, thermal-, and ultrasound-sensitive designs—which allow spatiotemporal control over growth factor release. The platform applications are organized by tissue types, encompassing soft tissue regeneration, bone and cartilage repair, neuroregeneration, cardiovascular regeneration, wound healing, anti-fibrotic therapies, and cancer microenvironment modulation. Each section provides recent case studies demonstrating how heparin enhances the bioactivity, localization, and therapeutic efficacy of pro-regenerative or anti-pathologic growth factors. Collectively, these insights highlight heparin’s dual role as both a carrier and modulator, positioning it as a pivotal component in next-generation, precision-targeted delivery systems. Full article

Background/Objectives: Obesity and type 2 diabetes mellitus (T2DM) profoundly disrupt lipid metabolism within local microenvironments of skeletal muscle and its associated connective tissues, including adipose tissue, bone, and fascia. However, the role of local communication between skeletal muscle and its proximal connective tissues in propagating metabolic dysfunction is incompletely understood. This narrative review synthesizes current evidence on these local metabolic interactions, highlighting novel insights and existing gaps. Methods: We conducted a comprehensive literature analysis of primary research published in the last decade, sourced from PubMed, Web of Science, and ScienceDirect. Studies were selected for relevance to skeletal muscle, adipose tissue, fascia, and bone lipid metabolism in the context of obesity and T2DM, with emphasis on molecular, cellular, and paracrine mechanisms of local crosstalk. Findings were organized into thematic sections addressing physiological regulation, pathological remodeling, and inter-organ signaling pathways. Results: Our synthesis reveals that local lipid dysregulation in obesity and T2DM involves altered fatty acid transporter dynamics, mitochondrial overload, fibro-adipogenic remodeling, and compartment-specific adipose tissue dysfunction. Crosstalk via myokines, adipokines, osteokines, bioactive lipids, and exosomal miRNAs integrates metabolic responses across these tissues, amplifying insulin resistance and lipotoxic stress. Emerging evidence highlights the underappreciated roles of fascia and marrow adipocytes in regional lipid handling. Conclusions: Collectively, these insights underscore the pivotal role of inter-tissue crosstalk among skeletal muscle, adipose tissue, bone, and fascia in orchestrating lipid-induced insulin resistance, and highlight the need for integrative strategies that target this multicompartmental network to mitigate metabolic dysfunction in obesity and T2DM. Full article

Background: Fixation influences the quality of staining across species, especially in neuroscience, where accurate visualization of neuronal structures and protein localization is crucial for understanding brain function and pathology. This study compared two commonly used fixatives—9% glyoxal (G-fix) and Davidson’s solution (D-fix)—regarding their effects on autofluorescence, immunolabeling specificity, and histological quality in medaka brain tissue. Methods: Mixed-sex medaka from five strains were fixed with either G-fix or D-fix. Autofluorescence was assessed in posterior bodies and brain tissues, including sections stained with fluorescently conjugated secondary antibodies alone. Tissues were also injected with fluorescent dyes or immunolabeled for neuronal markers (PGP9.5, NeuN, and NCAM) using fluorescent secondary antibodies. Hematoxylin and eosin (H&E) staining and immunohistochemistry were used to evaluate tissue morphology and chromogenic antigen detection. Results: Both fixatives induced autofluorescence: D-fix enhanced blue signals, while G-fix increased green and red fluorescence. These autofluorescence levels were significantly weaker than those from fluorescent dyes or PGP9.5 immunolabeling. Posterior body tissue showed patterns similar to deparaffinized brain sections, supporting its use for pre-screening fixation. G-fix yielded more neuron-specific PGP9.5 staining, whereas D-fix showed broader signal distribution. NeuN and NCAM were not detected, likely due to antibody incompatibility. PGP9.5 was undetectable by immunohistochemistry, while D-fix provided superior H&E staining quality. Conclusions: Although both fixatives induced autofluorescence, their signals were weaker than those of conventional dyes and antibodies. Glyoxal improved specificity for neuronal immunofluorescence, while Davidson enhanced histological detail. These findings provide practical guidance for optimizing fixation strategies in medaka-based neuroscience and histopathological research. Full article

Pelvic inflammatory disease (PID) encompasses a broad range of infection-induced inflammatory disorders of the female upper genital tract, commonly caused by ascending sexually transmitted infections. Diagnosis is often challenging because of nonspecific or absent symptoms and the overlap with other pelvic pathologies. While clinical and laboratory assessments are essential, cross-sectional imaging plays a pivotal role, especially in complicated, atypical, or equivocal cases. This review focuses on the typical and atypical imaging features of PID and highlights the crucial roles of computed tomography (CT) and magnetic resonance imaging (MRI) in its diagnostic evaluation. CT is frequently employed in emergency settings because of its widespread availability and ability to detect acute complications such as tubo-ovarian abscesses (TOA), peritonitis, or Fitz-Hugh–Curtis syndrome. However, it is limited by ionizing radiation and suboptimal soft-tissue contrast. MRI provides superior tissue characterization and multiplanar imaging without radiation exposure. When combined with diffusion-weighted imaging (DWI), MRI achieves high diagnostic accuracy, particularly in differentiating PID from other entities such as endometriosis, adnexal tumors, and gastrointestinal or urinary tract diseases. This review also addresses PID in specific clinical contexts, including post-partum infection, post-assisted reproductive technologies (ART), intrauterine device (IUD) use, and chronic or recurrent forms. A comprehensive, multimodal imaging approach integrated with clinical findings is essential for timely diagnosis, effective treatment, and prevention of severe reproductive sequelae. Full article

Background/Objectives: In recent years, lipids have emerged as critical regulators of different disease processes, being involved in cancer pathogenesis, progression, and outcome. Matrix-Assisted Laser Desorption/Ionization Mass Spectrometry Imaging (MALDI-MSI) has significantly expanded the technology’s reach, enabling spatially resolved profiling of lipids directly from tissue, including formalin-fixed paraffin-embedded (FFPE) specimens. In this context, MALDI matrix selection is crucial for lipid extraction and ionization, influencing key aspects such as molecular coverage and sensitivity, especially in such specimens with already depleted lipid content. Thus, in this work, we aim to explore the feasibility of mapping lipid species in FFPE clinical samples with MALDI-MSI using 6-aza-2-thiothymine (ATT) as a matrix of choice. Methods: To do so, ATT performances were first compared to those two other matrices commonly used for lipidomic analyses, 2′,5′-dihydroxybenzoic acid (DHB) and Norharmane (NOR), on lipid standards. Results: As a proof-of-concept, we then assessed ATT’s performance for the MALDI-MSI analysis of lipids in FFPE brain sections, both in positive and negative ion modes, comparing results with those obtained from other commonly used dual-polarity matrices. In this context, ATT enabled the putative annotation of 98 lipids while maintaining a well-balanced detection of glycerophospholipids (60.2%) and sphingolipids (32.7%) in positive ion mode. It outperformed both DHB and NOR in the identification of glycolipids (3%) and fatty acids (4%). Additionally, ATT exceeded DHB in terms of total lipid count (62 vs. 21) and class diversity and demonstrated performance comparable to NOR in negative ion mode. Moreover, ATT was applied to a FFPE glioblastoma tissue microarray (TMA) evaluating the ability of this matrix to reveal biologically relevant lipid features capable of distinguishing normal brain tissue from glioblastoma regions. Conclusions: Altogether, the results presented in this work suggest that ATT is a suitable matrix for pathology imaging applications, even at higher lateral resolutions of 20 μm, not only for proteomic but also for lipidomic analysis. This could enable the use of the same matrix type for the analysis of both lipids and peptides on the same tissue section, offering a unique strategic advantage for multi-omics studies, while also supporting acquisition in both positive and negative ionization modes. Full article

Scaffolds are widely used in bioengineering, both as 3D native tissue-mimicking models for investigating mechanisms under physiological and pathological conditions and also as implantable agents in regenerative medicine. Histological approaches, mainly formalin-fixed paraffin-embedded (FFPE) and frozen sample sectioning, are commonly applied to evaluate cell distribution and tissue-like properties of scaffolds. However, standard histological processing is not always compatible with the materials that scaffolds are made of. Thus, some adaptations to protocols are required to obtain intact sections. In this review we discuss challenges related to the histological processing of scaffolds and solutions to overcome them. We sequentially cover processing steps of the three main histological techniques for sample preparation—cryomicrotomy, FFPE samples microtomy and vibrating microtomy. Furthermore, we highlight the critical considerations in choosing the most appropriate method based on scaffold composition, mechanical properties and the specific research question. The goal of this review is to provide practical guidance on choosing reliable histological evaluation of complex scaffold-based systems in tissue engineering research. Full article

Background/Objectives: Prostate cancer (PCa) remains a major global health issue, associated with significant mortality and morbidity. Despite advances in diagnosis and treatment, predicting biochemical recurrence (BCR) after radical prostatectomy remains challenging, highlighting the need for reliable biomarkers to guide prognosis and therapy. The study aimed to evaluate the prognostic significance of the PTEN and ERG biomarkers in predicting BCR and tumor progression in PCa patients who underwent radical prostatectomy. Methods: This study consisted of a cohort of 91 patients with localized PCa who underwent radical prostatectomy between 2016 and 2022. From this cohort, 77 patients were selected for final analysis. Tissue microarrays (TMAs) were constructed from paraffin blocks, and immunohistochemical (IHC) staining for PTEN and ERG was performed using specific antibodies on the Ventana BenchMark ULTRA system (Roche Diagnostics, Indianapolis, IN, USA). Stained sections were evaluated and correlated with clinical and pathological data. Results: PTEN expression showed a significant negative correlation with BCR (r = −0.301, p = 0.014), indicating that reduced PTEN expression is associated with increased recurrence risk. PTEN was not significantly linked to PSA levels, tumor stage, or lymph node involvement. ERG expression correlated positively with advanced pathological tumor stage (r = 0.315, p = 0.005) but was not associated with BCR or other clinical parameters. Conclusions: PTEN appears to be a valuable prognostic marker for recurrence in PCa, while ERG may indicate tumor progression. These findings support the potential integration of PTEN and ERG into clinical practice to enhance risk stratification and personalized treatment, warranting further validation in larger patient cohorts. Full article

Background/Objective: The Rs1 exon-1-del rat (Rs1KO) XLRS model shows normal retinal development until postnatal day 12 (P12) when small cystic spaces start to form in the inner nuclear layer. These enlarge rapidly, peak at P15, and then collapse by P19. These events overlap with eye opening at P12–P15. We investigated whether new light-driven retinal activity could contribute to the appearance and progression of schisis cavities in this rat model of XLRS disease. Methods: For dark rearing (D/D), mating pairs of Rs1KO strain were raised in total darkness in a special vivarium at UC Davis. When pups were born, they were maintained in total darkness, and eyes were collected at P12, P15, and P30 (n = 3/group) for each of the D/D and cyclic light-reared 12 h light–12 h dark (L/D) Rs1KO and wild-type (WT) littermates. Eyes were fixed, paraffin-embedded, and sectioned. Tissue morphology was examined by H&E and marker expression of retinoschisin1 (Rs1), rhodopsin (Rho), and postsynaptic protein 95 (Psd95) by fluorescent immunohistochemistry. H&E-stained images were analyzed with ImageJ version 1.54h to quantify cavity size using the “Analyze Particles” function. Results: Small intra-retinal schisis cavities begin to form by P12 in the inner retina of both D/D and L/D animals. Cavity formation was equivalent or more pronounced in D/D animals than in L/D animals. We compared Iba1 (activation marker of immune cells) distribution and found that by P12, when schisis appeared, Iba1⁺ cells had accumulated in regions of schisis. Iba1⁺ cells were more abundant in Rs1KO animals than WT animals and appeared slightly more prevalent in D/D- than L/D-reared Rs1KO animals. We compared photoreceptor development using Rho, Rs1, and Psd95 expression, and these were similar; however, the outer segments (OSs) of D/D animals with Rho labeling at P12 were longer than L/D animals. Conclusions: The results showed that cavities formed at the same time in D/D and L/D XLRS rat pups, indicating that the timing of schisis formation is not light stimulus-driven but rather appears to be a result of developmental events. Cavity size tended to be larger under dark-rearing conditions in D/D animals, which could be due to the decreased rate of phagocytosis by the RPE in the dark, allowing for continued growth of the OSs without the usual shedding of the distal tip, a key mechanism behind dark adaptation in the retina. These results highlight the complexity of XLRS pathology; however, we found no evidence that light-driven metabolic activity accounted for schisis cavity formation. Full article

Epilepsy is one of the most prevalent chronic medical conditions that really can affect individuals at any age. A broader study of the pathogenesis of the epileptic condition will probably serve as the cornerstone for the development of new antiepileptic remedies that aim to treat epilepsy symptomatically as well as prevent the epileptogenesis process or regulate its progression. Cellular changes in the brain include oxidative stress, neuroinflammation, inflammatory cell invasion, angiogenesis, and extracellular matrix associated changes. The extensive molecular profiling of epileptogenic tissue has revealed details on the molecular pathways that might start and sustain cellular changes. In healthy brains, epilepsy develops because of vascular disruptions, such as blood–brain barrier permeability and pathologic angiogenesis. Key inflammatory mediators are elevated during epileptic seizures, increasing the risk of recurrent seizures and resulting in secondary brain injury. Prostaglandins and cytokines are well-known inflammatory mediators in the brain and, after seizures, their production is increased. These inflammatory mediators may serve as therapeutic targets in the clinical research of novel antiepileptic medications. The functions of inflammatory mediators in epileptogenesis are covered in this review. Oxidative stress also plays a significant role in the pathogenesis of various neurological disorders, specifically epilepsy. Antioxidant therapy seems to be crucial for treating epileptic patients, as it prevents neuronal death by scavenging excess free radicals formed during the epileptic condition. The significance of antioxidants in mitochondrial dysfunction prevention and the relationship between oxidative stress and inflammation in epileptic patients are the major sections covered in this review. Full article

Objectives: This study aimed to determine the factors affecting the diagnostic efficacy of frozen sections for assessing tumor spread through air spaces (STAS) and to provide suggestions for the sampling of these frozen sections. Methods: Cases of invasive adenocarcinoma with a pathological diagnosis of stage IA-IIIB were screened, and frozen and paraffin sections were reviewed. Using paraffin sections as the gold standard, the consistency of frozen pathological diagnosis of STAS was calculated. Factors that may affect STAS diagnosis in frozen sections were screened, and a nomogram was drawn. Results: The sensitivity of frozen sections in STAS diagnosis was 55.4% (108/195), the specificity was 74.5% (254/321), and the kappa value was 0.35. In the subsequent logistic regression, the ratio of the tissue diameters of the frozen and paraffin sections, number of frozen section sheets, clarity of the tumor boundary, and number of alveoli from the peritumoral area to tissue edge were all statistically significantly significant (p < 0.05) factors affecting the frozen STAS diagnostic efficacy. Conclusions: The diagnostic efficacy of frozen sections for STAS is poor. In our study, the tissue diameter ratio of the frozen to paraffin sections, the number of frozen sections, the clarity of the tumor boundary, and the number of alveoli from the peritumoral tissue to the tissue edge were considered independent factors affecting diagnostic consistency. The accuracy of the frozen section analysis in STAS diagnosis can be improved by our reasonable suggestions on frozen sampling, making it a reliable indicator of the surgical method. Full article