Search Results (159)

This study investigated the interactive effects of dietary vitamin A (VA) and all-trans retinoic acid (ATRA) on growth performance and intestinal health in broilers. A total of 432 one-day-old male Arbor Acres chicks were assigned to a 2 × 3 factorial design with two VA levels (2000 and 6000 IU/kg) and three ATRA levels (0, 0.25, and 0.50 mg/kg). The maize–soybean meal basal diet contained 180 IU/kg VA without extra VA supplementation. Results showed that compared with 0 mg/kg ATRA, 0.50 mg/kg ATRA enhanced average daily gain (ADG) during days 1–21 (p < 0.05). Compared with 2000 IU/kg VA, 6000 IU/kg VA improved body weight on day 35 as well as ADG and feed intake during days 22–35 and reduced feed conversion ratio over the entire trial (p < 0.05). There were VA × ATRA interactions for the ratio of villus height (VH) to crypt depth (CD) in duodenum as well as VH and CD in ileum on day 21 (p < 0.05). The 0.25 mg/kg ATRA decreased duodenal VH/CD and ileal VH in broilers fed 2000 and 6000 IU/kg VA, respectively (p < 0.05). The 0.50 mg/kg ATRA increased ileal VH in broilers fed both 2000 and 6000 IU/kg VA (p < 0.05). When birds were fed 6000 IU/kg VA, 0.50 mg/kg ATRA increased ileal CD compared with 0.25 mg/kg CD (p < 0.05). On day 35, compared with 0 mg/kg ATRA, 0.25 mg/kg ATRA increased ileal VH while 0.50 mg/kg ATRA decreased ileal CD, and both of them increased ileal VH/CD (p < 0.05). The VA × ATRA interactions for mRNA expression of jejunal Mucin5ac on day 21 and jejunal Occludin, Claudin-1, Mucin 2, leucine-rich-repeat-containing G-protein-coupled receptor 5⁺ (Lgr5⁺), zinc and ring finger 3 (Znrf3), and secreted phosphoprotein 1 (SPP1) on day 35 were detected (p < 0.05). Dietary 0.50 mg/kg ATRA up-regulated jejunal Mucin5ac expression in broilers fed 6000 IU/kg VA on day 21 as well as Claudin-1, Znrf3, and SPP1 expression broilers fed 2000 IU/kg VA on day 35 (p < 0.05). The 0.25 mg/kg ATRA down-regulated Occludin expression in broilers fed 6000 IU/kg VA on day 35 (p < 0.05). The 0.25 mg/kg ATRA decreased and increased Lgr5⁺ expression on day 35 in broilers fed 2000 and 6000 IU/kg VA, respectively (p < 0.05). Both 0.25 and 0.50 mg/kg ATRA down-regulated Mucin-2 expression in broilers fed 2000 IU/kg VA on day 35 (p < 0.05). The VA × ATRA interactions were observed for jejunal retinol dehydrogenase 10 (RDH10), cytochrome P450, family 26, subfamily A, polypeptide 1 (CYP26A1), retinoic acid receptor (RAR) α, and RARβ expression on days 21 and 35 (p < 0.05). Both 0.25 and 0.50 mg/kg up-regulated RDH10, CYP26A1, and RARβ expression in broilers fed 6000 IU/kg VA (p < 0.05). The RARα expression was up-regulated by 0.50 and 0.25 mg/kg ATRA on days 21 and 35, respectively (p < 0.05). Plasma metabolomics identified 269 VA- and 185 ATRA-associated differential metabolites, primarily enriched in lipid metabolism, vitamin digestion and absorption, and bacterial infection pathways. In conclusion, dietary 0.50 mg/kg ATRA and 6000 IU/kg VA enhanced growth performance, intestinal integrity, and VA metabolism, partly through activation of retinoic acid receptors and modulation of plasma lipid metabolism. Full article

SHORT INTERNODE (SHI)-Related Sequence (SRS) transcription factors play crucial roles in plant growth, development, and stress responses and have been extensively studied in various plant species. However, the molecular functions and regulatory mechanisms of SRS genes in the economically important tropical fruit crop pitaya (Hylocereus undatus) remain poorly understood. This study identified 9 HuSRS genes in pitaya via bioinformatics analysis, with subcellular localization predicting nuclear distributions for all. Gene structure analysis showed 1–4 exons, and conserved motifs (RING-type zinc finger and IXGH domains) were shared across subclasses. Phylogenetic analysis classified the HuSRS genes into three subfamilies. Subfamily I (HuSRS1–HuSRS4) is closely related to poplar and tomato homologs and subfamily III (HuSRS6–HuSRS8) contains a recently duplicated paralogous pair (HuSRS7/HuSRS8) and shows affinity to rice SRS genes. Protein structure prediction revealed dominance of random coils, α-helices, and extended strands, with spatial similarity correlating to subfamily classification. Interaction networks showed HuSRS1, HuSRS2, HuSRS7 and HuSRS8 interact with functional proteins in transcription and hormone signaling. Promoter analysis identified abundant light/hormone/stress-responsive elements, with HuSRS5 harboring the most motifs. Transcriptome and qPCR analyses revealed spatiotemporal expression patterns: HuSRS4, HuSRS5, and HuSRS7 exhibited significantly higher expression levels in callus (WG), which may be associated with dedifferentiation capacity. In seedlings, HuSRS9 exhibited extremely high transcriptional accumulation in stem segments, while HuSRS1, HuSRS5, HuSRS7 and HuSRS8 were highly active in cotyledons. This study systematically analyzed the characteristics of the SRS gene family in pitaya, revealing its evolutionary conservation and spatio-temporal expression differences. The research results have laid a foundation for in-depth exploration of the function of the SRS gene in the tissue culture and molecular breeding of pitaya. Full article

The ring finger protein 213 (RNF213) Arg4810Lys variant has been previously identified as a significant risk factor for Moyamoya disease (MMD), particularly in East Asian populations. This review explores the broader impact of the Arg4810Lys mutation on various cerebrovascular conditions, including Moyamoya syndrome (MMS), intracranial artery stenosis, quasi-Moyamoya syndromes, ischemic stroke, and intracranial atherosclerosis. Beyond the brain, it is also implicated in pulmonary arterial hypertension, coronary artery disease, and renal artery stenosis, emphasizing its systemic effects. Functional studies suggest that RNF213 Arg4810Lys alters angiogenic signaling, endothelial cell function, vascular remodeling, and immune response pathways, especially when influenced by environmental stressors, like hypoxia or inflammation. The gene dosage of Arg4810Lys significantly affects disease phenotypes, with homozygous carriers typically experiencing earlier onset with increased severe symptoms. The variant also exhibits incomplete penetrance and frequently co-occurs with additional genetic alterations, including trisomy, KIF1A, FLNA, and PCSK9 mutations, which complicates its pathogenicity. A comprehensive understanding of RNF213 Arg4810Lys’s systemic impact is essential to developing effective risk assessment strategies, personalized treatments, and targeted therapies for associated vascular diseases. Full article

Swine acute diarrhea syndrome coronavirus (SADS-CoV) is a recently discovered enteric coronavirus that has caused considerable economic losses in the pig industry. SADS-CoV was first reported in 2017 in Guangdong Province, China, and subsequently in Fujian, Guangxi, Henan and Jiangxi Provinces. Bone marrow stromal cell antigen 2 (BST-2), also known as tetherin, acts as an antiviral protein to limit the release of a wide range of enveloped viruses. However, the relationship between BST-2 and SADS-CoV has rarely been studied. Here, we showed that endogenous BST-2 expression is downregulated by SADS-CoV infection in Vero-E6 and ST cells by 2- to 3-fold. The overexpression of BST-2 inhibited SADS-CoV replication, whereas the knockdown of the BST-2 gene in Vero cells restored SADS-CoV replication. Further study revealed that BST-2 targets the SADS-CoV nucleocapsid protein (N) and decreases N protein expression, and that the BST-2 transmembrane (TM) domain is essential for this activity. Moreover, the degradation of the SADS-CoV N protein promoted by BST-2 is mediated by the membrane-associated ring-CH-type finger 8 (MARCHF8)/calcium binding and coiled-coil domain 2 (NDP52) autophagosome pathway. Overall, we found that BST-2 suppresses viral proliferation by inducing the breakdown of the SADS-CoV N protein via the MARCHF8/NDP52 pathway. Full article

Sarcopenia is characterized by a reduction in muscle function and skeletal muscle mass relative to that of healthy individuals. In older adults and those who are less resistant to sarcopenia, glucocorticoid secretion or accumulation during treatment exacerbates muscle protein degradation, potentially causing sarcopenia. This study assessed the preventive effects and mechanisms of heat-killed Lactobacillus plantarum postbiotic beLP-K (beLP-K) against dexamethasone (DEX)-induced sarcopenia in C2C12 myotubes and Sprague-Dawley rats. The administration of beLP-K did not induce cytotoxicity and mitigated cell damage caused by DEX. Furthermore, beLP-K significantly reduced the expression of forkhead box O3 α (FoxO3α), muscle atrophy f-box (MAFbx)/atrogin-1, and muscle RING-finger protein-1 (MuRF1), which are associated with muscle protein degradation. DEX induced weight loss in rats; however, in the beLP-K group, weight gain was observed. Micro-computed tomography analysis revealed that beLP-K increased muscle mass, correlating with weight and grip strength. beLP-K alleviated the DEX-induced reduction in grip strength and increased the mass of hind leg muscles. The correlation between beLP-K administration and increased muscle mass was associated with decreased expression levels of muscle degradation-related proteins such as MAFbx/atrogin-1 and MuRF1. Therefore, beLP-K may serve as a treatment for sarcopenia or as functional food material. Full article

We identified Murashka, a RING finger protein, in an oogenesis screen as a regulator of Drosophila ovary germline stem cell niche development. Mutant alleles of murashka exhibited an enlarged niche phenotype reminiscent of increased Notch signalling and displayed genetic interactions with Notch alleles, and with polychaetoid, a regulator of Notch during niche development. These interactions uncovered both positive and negative impacts on Notch in different genetic backgrounds. In S2 cells, Murashka formed a complex with Notch and colocalised with Notch in the secretory pathway. Murashka expression in S2 cells down-regulated Notch signalling levels but could result in increased fold induction due to the proportionally greater decrease in basal ligand-independent activity. In vivo Murashka expression had different outcomes on different Notch target genes. We observed a decrease in the expression of vestigial along the anterior/posterior boundary of the wing imaginal disc, but not of wingless at the dorsal/ventral boundary. Instead, weak ectopic wingless was observed, which was synergistically increased by the coexpression of Deltex, a positive regulator of ligand-independent signalling. Our results identify a novel developmental role for murashka, a gene previously only associated with a function in long-term memory, and indicate a regulatory role for Murashka through a physical interaction with Notch that has context-dependent outcomes. Murashka adds to a growing number of ubiquitin ligase regulators which interact with Notch at different locations within its secretory and endocytic trafficking pathways. Full article

Background/Objectives: Sarcopenia is an age-related disease resulting in muscle mass deterioration and declining strength and functional ability. Muscle protein degradation pathways are activated through the ubiquitin–proteasome system, which is integral to the pathogenesis of sarcopenia. This study examined the capability of Lactobacillus plantarum beLP1 as a postbiotic ingredient of kimchi that prevents sarcopenia. Methods: We evaluated cell viability and measured diameters in a C2C12 myotube damage model and muscle volume, muscle weight, muscle strength, and the expression of muscle degradation proteins MuRF1 and Atrogin-1 in dexamethasone-induced sarcopenic model rats using a heat-killed beLP1 strain. Results: beLP1 had no cytotoxic effects on C2C12 and prevented dexamethasone-induced cellular damage, suggesting its role in muscle protein degradation pathways. beLP1 treatment significantly prevented the dexamethasone-induced reduction in myotube diameter. In a dexamethasone-induced sarcopenic rat model, oral beLP1 significantly mitigated muscle mass decline and prevented grip strength reduction. Microcomputed tomography demonstrated that beLP1 reduced dexamethasone-induced muscle volume loss. beLP1 treatment reduced Atrogin-1 and Muscle RING-finger protein-1 (MuRF1) and the transcription factor Forkhead box O3 alpha (FoxO3α), which triggers muscle protein breakdown. beLP1 exerts protective effects by inhibiting the ubiquitin-proteasome system and regulating FoxO3α signaling. It increased AKT (Ser473) phosphorylation, which affected muscle protein synthesis, degradation, and cell survival, suggesting its potential to prevent sarcopenia. Conclusions: Heat-killed Lactobacillus plantarum beLP1 alleviates muscle mass wasting and weakness in a dexamethasone-induced sarcopenia model by regulating muscle protein degradation pathways and signaling molecules. Thus, postbiotics may be functional ingredients in sarcopenia prevention. Full article

RNA-binding E3 ubiquitin ligases (RBULs) provide a link between RNA metabolic processes and the ubiquitin proteasome system (UPS). In humans, RBULs are involved in various biological processes, such as cell proliferation and differentiation, as well as sexual development. To date, little is known about their role in the protozoan parasite Plasmodium falciparum, the causative agent of malaria tropica. We previously identified a novel P. falciparum RBUL, the RING finger E3 ligase PfRNF1, which is highly expressed during gametocyte development. Here, we conducted BioID-based proximity interaction studies to unveil the PfRNF1 interactome. We show that in immature gametocytes, PfRNF1 forms an interaction network that is mainly composed of RNA-binding proteins, including the translational repressors DOZI and CITH and members of the CCR4-NOT complex, as well as UPS-related proteins. In particular, PfRNF1 interacts with recently identified regulators of sexual development like the zinc finger protein PfMD3, with which it shares the majority of interactors. The common interactome of PfRNF1 and PfMD3 comprises several uncharacterized proteins predominantly expressed in male or female gametocytes. Our results demonstrate that PfRNF1 engages with RNA-binding proteins crucial for sex determination in gametocytes, thereby linking posttranscriptional regulation with the UPS. Full article

This overview explores the complex relationship between environmental factors, particularly obesity, and the timing of puberty, with a focus on how hormonal and genetic interactions are influenced by external conditions. Puberty (gonadarche) is characterised by the activation of the hypothalamic–pituitary–gonadal (HPG) axis. The onset and progression of puberty vary significantly among individuals, primarily due to genetic factors, with key genes like kisspeptin 1 (KISS1) and makorin ring finger protein 3 (MKRN3) playing a crucial role. Cohesively, this paper emphasises that environmental factors, particularly obesity and exposure to endocrine-disrupting chemicals (EDCs), have become significant influences on the timing of puberty. Childhood obesity has risen significantly in recent decades and the age of pubertal onset has declined over the same period. Obesity greatly disrupts hormone regulation in pre-pubertal children. Leptin accelerates the onset of puberty in girls but not in boys. The underlying mechanism is proposed to be the increase in Kiss1/GnRH signalling. On the contrary, excess leptin in boys suppresses testosterone production by increasing oestrogen conversion. Low adiponectin in obese girls may contribute to earlier puberty due to a reduced inhibition of Kiss1/GnRH signalling. Low adiponectin in boys is linked to delayed puberty due to its role in maintaining insulin sensitivity and testosterone production. Hyperinsulinemia influences pubertal timing through central and peripheral mechanisms. Insulin acting synergistically with leptin promotes the earlier onset of puberty in girls but not in boys. The effects of exposure to certain EDCs—mostly obesogenic chemicals that mimic the action of natural hormones—on the timing of puberty remain unclear; hence, further research on this topic is needed. Addressing and preventing obesity in children could potentially mitigate these alterations in pubertal timing. Full article

MuRF1 [muscle RING (Really Interesting New Gene)-finger protein-1] is an ubiquitin-protein ligase (E3), which encode by TRIM63 (tripartite motif containing 63) gene, playing a crucial role in regulating cardiac muscle size and function through ubiquitylation. Among hypertrophic cardiomyopathy (HCM) patients, 24 TRIM63 variants have been identified, with 1 additional variant linked to restrictive cardiomyopathy. However, only three variants have been previously investigated for their functional effects. The structural impacts of the 25 variants remain unexplored. This study investigated the effects of 25 MuRF1 variants on ubiquitylation activity using in vitro ubiquitylation assays and structural predictions using computational approaches. The variants were generated using site-directed PCR (Polymerase Chain Reaction) mutagenesis and subsequently purified with amylose affinity chromatography. In vitro ubiquitylation assays demonstrated that all 25 variants compromised the ability of MuRF1 to monoubiquitylate a titin fragment (A168-A170), while 17 variants significantly impaired or completely abolished auto-monoubiquitylation. Structural modelling predicted that 10 MuRF1 variants disrupted zinc binding or key stabilising interactions, compromising structural integrity. In contrast, three variants were predicted to enhance the structural stability of MuRF1, while six others were predicted to have no discernible impact on the structure. This study underscores the importance of functional assays and structural predictions in evaluating MuRF1 variant pathogenicity and provides novel insights into mechanisms by which these variants contribute to HCM and related cardiomyopathies. Full article

Muscle atrophy leads to decreased muscle mass, weakness, inactivity, and increased mortality. E3 ubiquitin ligases, key regulators of protein degradation via the ubiquitin–proteasome system, play a critical role in atrophic mechanisms. This meta-analysis followed Preferred Reporting Items for Systematic reviews and Meta-Analyses (PRISMA) guidelines, and its objective was to evaluate the association between E3 ligases Muscle Atrophy F-box (MAFbx)/Atrogin-1 (Fbxo32) and Muscle RING-finger protein 1 (MuRF-1) (TRIM63) E3 ligase mRNA levels, reductions in skeletal muscle CSA measures, and atrophy conditions. We examined papers published on PubMed^®, Scopus, and Web of Science that studied E3 ligase gene expression signatures for Fbxo32 (MAFbx/Atrogin-1) and Trim63 (MuRF1) in different types of muscle atrophy and hypertrophy murine models. Twenty-nine studies selected by two independent raters were analyzed. Standardized mean differences (SMDs)/effect sizes (ESs) and 95% confidence intervals (CIs) were calculated for the outcomes using fixed-effects models. We found that 6- and 4.8-fold upregulation, respectively, of Fbxo32 and Trim63 was sufficient to reduce the ES to −3.89 (95% CI: −4.45 to −3.32) for the muscle fiber cross-sectional area and the development of skeletal muscle atrophy. I² and Q test statistics did not indicate heterogeneous data. There was a low probability of bias after both the funnel plot and Egger’s test analyses. These results were sustained independently of the atrophic model and muscle type. Therefore, the magnitude of the increase in muscle Fbxo32 and Trim63 mRNA is a feasible, reliable molecular marker for skeletal muscle atrophy in mice. The next step for the Ubiquitin-proteasome system (UPS) field involves elucidating the targets of E3 ligases, paving the way for diagnostic and treatment applications in humans. Full article

Metabolic-associated fatty liver disease (MAFLD) is a chronic liver condition with limited therapeutic options. To identify novel drug targets, we integrated bioinformatics, Mendelian randomization (MR), and colocalization analyses. Using the Gene Expression Omnibus (GEO) database, we identified differentially expressed genes and constructed protein–protein interaction (PPI) networks, pinpointing 10 hub genes. MR and colocalization analyses revealed that Ubiquitin-like with PHD and ring finger domains 1 (UHRF1) is causally associated with MAFLD and driven by the same causal variant locus, suggesting its potential as a therapeutic target. Molecular docking identified disogenin as a candidate small-molecule drug targeting UHRF1. Drug affinity responsive target stability (DARTS) assays confirmed direct binding between UHRF1 and disogenin. In vitro, disogenin significantly reduced UHRF1 mRNA and protein levels induced by free fatty acids (FFA) in AML12 and HepG2 cells, accompanied by decreased cellular total cholesterol (TC) and triglyceride (TG) levels. In vivo, disogenin administration alleviated hepatic lipid accumulation, inflammation, and fibrosis in methionine/choline-deficient (MCD)-diet-fed mice. This study identifies UHRF1 as a promising therapeutic target for MAFLD and validates disogenin as a potential therapeutic agent, providing a foundation for further investigation. Full article

B30.2 domains, sometimes referred to as PRY/SPRY domains, were originally identified by sequence profiling methods at the gene level. The B30.2 domain comprises a concanavalin A-like fold consisting of two twisted seven-stranded anti-parallel β-sheets. B30.2 domains are present in about 150 human and 700 eukaryotic proteins, usually fused to other domains. The B30.2 domain represents a scaffold, which, through six variable loops, binds different unrelated peptides or endogenous low-molecular-weight compounds. At the cellular level, B30.2 proteins engage in supramolecular assemblies with important signaling functions. In humans, B30.2 domains are often found in E3-ligases, such as tripartite motif (Trim) proteins, SPRY domain-containing SOCS box proteins, Ran binding protein 9 and −10, Ret-finger protein-like, and Ring-finger proteins. The B30.2 protein recognizes the target and recruits the E2-conjugase by means of the fused domains, often involving specific adaptor proteins. Further well-studied B30.2 proteins are the methyltransferase adaptor protein Ash2L, some butyrophilins, and Ryanodine Receptors. Although the affinity of an isolated B30.2 domain to its ligand might be weak, it can increase strongly due to avidity effects upon recognition of oligomeric targets or in the context of macromolecular machines. Full article

Diabetic kidney disease (DKD) is a prevalent complication associated with diabetes in which podocyte dysfunction significantly contributes to the development and progression of the condition. Ring finger protein 183 (RNF183) is an ER-localized, transmembrane ring finger protein with classical E3 ligase activity. However, whether RNF183 is involved in glomerular podocyte dysfunction, which is the mechanism of action of DKD, is still poorly understood. In this study, we first demonstrated that RNF183 expression in glomerular podocytes of patients with DKD decreased as the disease progressed. Additionally, our transcriptome sequencing analysis of kidney tissues from diabetic mice revealed a significant reduction in RNF183 expression within the kidney cortex. Similarly, the expression of RNF183 was significantly reduced both in the kidneys of diabetic mice and in human podocytes exposed to high glucose conditions. The downregulation of RNF183 resulted in a suppression of autophagic activity, an increase in apoptotic cell death, and reduced expression of cellular markers in HPC cells. We found that RNF183 was modified via N6-methyladenosine (m6A) RNA methylation. Meanwhile, treatment with meclofenamic acid 2 (MA2), an m6A demethylase inhibitor, resulted in the upregulation of RNF183 expression in HPC cells cultured in high glucose conditions. Furthermore, high glucose treatment decreased the transcription and protein levels in both the m6A writer methyltransferaselike3 (METTL3) and the m6A reader insulin-like growth factor 2 mRNA-binding protein 2 (IGF2BP2). IGF2BP2 assisted with METTL3, which is jointly involved in the transcription of RNF183. Furthermore, we confirmed that RNF183 directly ubiquitinates M2 pyruvate kinase (PKM2) through co-immunoprecipitation (Co-IP) and liquid chromatography–mass spectrometry (LC-MS) experiments. The level of PKM2 ubiquitination was increased following RNF183 overexpression, leading to enhanced PKM2 protein degradation and subsequently alleviating high glucose-induced podocyte damage. The results of this study indicated that RNF183 was regulated via m6A methylation modification and that RNF183 expression was reduced in HPC cells treated with high glucose, which resulted in decreased PKM2 ubiquitination levels and subsequently aggravated podocyte injury. The findings suggest that RNF183 may serve as a potential therapeutic target for diabetic kidney injury, offering new insights into its role in the progression of DKD. Full article

Background/Objectives: Muscle-specific RING finger protein 1 (MuRF-1) is a pivotal regulator of muscle protein breakdown, an essential process for post-exercise muscle adaptation. This systematic review aimed to evaluate the effects of physical exercise on MuRF-1 mRNA expression in humans. Methods: A literature search was conducted in PubMed, Scopus, Cochrane Library, Google Scholar, and Web of Science following the PRISMA guidelines. The search was limited to studies published from 1 January 2001 to 1 December 2024. The inclusion and exclusion criteria were defined using the PICOS strategy. Two investigators independently performed the study selection, data extraction, and assessment of methodological quality, with any disagreements resolved by a third investigator. The PEDro scale was used to evaluate the risk of bias. Results: Forty-six studies met the eligibility criteria and were included. The findings evidenced that physical exercise significantly modulates MuRF-1 mRNA expression in humans. Resistance exercise induces transient increases, typically peaking between 1 and 4 h, whereas endurance exercise elicits similar responses within 40 min to 4 h post-exercise. Combined exercise protocols that include resistance and endurance exercises significantly increased MuRF-1 mRNA expression at 3 h post-exercise. The effects of physical exercise on MuRF-1 mRNA expression are influenced by factors such as exercise order, intensity, contraction mode, age, sex, and fitness level. Conclusions: This systematic review shows that MuRF-1 mRNA expression is significantly modulated by physical exercise in humans and is sensitive to different exercise modalities. These findings suggest that this key protein involved in muscle protein breakdown and turnover is essential for exercise-induced adaptations, contributing to skeletal muscle recovery and remodeling after exercise. Full article