Search Results (253)

Oxidative stress and inflammation are key drivers in the pathogenesis of various chronic diseases, including cardiovascular disease, neurodegenerative disorders, chronic kidney disease, and diabetes. This study evaluated the antioxidant and anti-inflammatory activities of SHE, CME, and FCME, all cultivated in northern Thailand. Human embryonic kidney cells (HEK293) were exposed to FeSO₄ to induce oxidative stress and to LPS to stimulate inflammation. Cell viability was assessed using the MTT assay, while intracellular ROS production was measured using the DCFH-DA. Lipid peroxidation was quantified using the thiobarbituric acid reactive substances assay, and the interleukin-6 (IL-6) release was determined by ELISAs. All extracts demonstrated low cytotoxicity; however, cell death increased at 48 h compared to 24 h. At 200 µg/mL, SHE, CME, and FCME significantly reduced the H₂O₂-induced ROS generation, with the combined treatment of SHE and FCME producing a more pronounced reduction than the individual treatments. Furthermore, the combination of SHE and FCME markedly decreased malondialdehyde (MDA) and IL-6 levels compared with other groups. These findings suggest that shallot and cha-miang extracts, particularly in combination, exhibit promising antioxidant and anti-inflammatory properties in kidney cell models. This combination could therefore be explored as a nutraceutical strategy for the prevention and management of chronic kidney disease, in which oxidative stress and inflammation play pivotal roles. Overall, our finding highlight the potential of the combined use of SHE and FCME as a functional ingredients in the food and pharmaceutical industries. Full article

Background/Objectives: SARS-CoV-2 continues to generate antigenically divergent variants that reduce the breadth of existing vaccine-induced antibody responses. Fc-fusion subunit vaccines offer advantages in stability, antigen display, and Fc-mediated immune engagement. This study aimed to design and evaluate a tetravalent RBD–Fc fusion construct incorporating RBDs from Wuhan-Hu-1 and Omicron BA.4/BA.5 and to determine whether this configuration can induce broad antibody recognition across SARS-CoV-2 variants. The objective was to assess its feasibility, biochemical properties, and initial immunogenicity. Methods: Immune responses to the construct were first assessed using the C-ImmSim simulation platform. The full-length fusion was synthesized, subcloned into pcDNA3.1(+), expressed in HEK293 cells, and purified by Protein G affinity chromatography. Protein integrity was evaluated by reducing SDS–PAGE. BALB/c mice (female, 8 weeks) were immunized with a prime–boost–boost schedule, and sera were analyzed by ELISA, considering binding to Wuhan-Hu-1, Omicron BA.4/BA.5, and a panel of RBD variants. Results: In silico analysis predicted coordinated antigen clearance, class switching, memory B- and CD4⁺ T-cell formation, and transient cytokine induction. The recombinant protein was expressed efficiently, yielding a major ~56 kDa band and a ~23 kDa RBD fragment. Vaccinated mice generated strong IgG responses to Wuhan-Hu-1 and BA.4/BA.5 RBDs and showed broad binding to major variant RBDs. Conclusions: The tetravalent RBD–Fc fusion vaccine was successfully produced and elicited broad antibody binding across SARS-CoV-2 variants, supporting its potential as a versatile protein-based vaccine platform. Full article

The production and quality of wheat and maize grain can be significantly affected by various pests and pathogens, with phytoplasmas posing a particular threat due to their rapid spread and potential to cause severe damage to cultivated crops. The objective of this investigation was to evaluate the risk associated with these wall-less bacteria in wheat and maize crops. To achieve this, a survey was conducted in commercial fields located in southwestern Poland. Samples of winter wheat and fodder maize were collected at two distinct developmental stages, including both symptomatic and asymptomatic plants. Symptoms observed in wheat included yellowing, stunting, and excessive tillering, while maize plants showed yellow leaf striping, red discoloration, and stunted growth. Polymerase chain reaction (PCR) assays using phytoplasma-specific primers, followed by Sanger sequencing and sequence analysis, confirmed phytoplasma infections in 2% of wheat and 1.5% of maize samples. Virtual restriction fragment length polymorphism (RFLP) analysis identified the wheat-infecting phytoplasmas as belonging to subgroup 16SrI-C (‘Candidatus Phytoplasma tritici’-related strain)—a pathogen of major concern for wheat, while maize-infecting phytoplasmas were classified into subgroups 16SrI-B and 16SrV-C. Additionally, wheat plants collected during the early elongation phase were tested for Mastrevirus hordei (former wheat dwarf virus, WDV) using double antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA), which confirmed the presence of WDV in all tested samples. Preliminary screening of field-collected leafhoppers revealed that 7.5% of Psammotettix alienus, the predominant species in wheat fields, carried 16SrI-C phytoplasmas. In maize fields, Zyginidia scutellaris was the most prevalent species, with 1.7% of individuals carrying 16SrV-C phytoplasma. These findings suggest that these insect species may contribute to the transmission of phytoplasmas in wheat and maize. This study provides the first documented evidence of 16SrI-C phytoplasma infecting wheat in Poland, and of 16SrV-C and 16SrI-B phytoplasmas infecting maize, expanding the known host range of these subgroups in the country and highlighting their potential phytosanitary importance. Full article

Background and Objectives: Pituitary adenomas are common intracranial tumors lacking specific non-invasive biomarkers. This study aimed to determine whether key metabolites and enzymes of the kynurenine pathway—including indoleamine 2,3-dioxygenase (IDO), kynurenine (KYN), kynurenic acid (KYNA), kynurenine aminotransferase (KAT), quinolinic acid, and picolinic acid—can serve as diagnostic biomarkers distinguishing patients with pituitary adenomas from healthy controls. Materials and Methods: We conducted a single-center, cross-sectional, case–control study with 50 patients with pituitary adenomas and 35 healthy controls. Serum levels of IDO, KYN, KYNA, KAT, quinolinic acid, and picolinic acid were measured via enzyme-linked immunosorbent assay (ELISA). Statistical analyses included group comparisons (t-test/Mann–Whitney U), multivariate logistic regression to identify independent predictors, receiver operating characteristic (ROC) curve analysis to evaluate diagnostic performance (area under the curve, AUC), and partial least squares discriminant analysis (PLS-DA) for multivariate metabolic profiling. Results: Serum kynurenine, kynurenic acid, 3-hydroxykynurenine, picolinic acid, IDO and kynureninase were significantly higher in the pituitary adenoma group than in healthy controls (p < 0.001), while tryptophan, kynurenine aminotransferase, anthranilic acid and quinolinic acid showed no significant differences. ROC analysis demonstrated excellent diagnostic accuracy, with KAT (AUC = 0.923) and KYNA (AUC = 0.901) showing the highest discrimination. Multivariate logistic regression identified IDO, KYN, and KYNA as independent predictors of pituitary adenoma (p < 0.05). PLS-DA of the combined metabolite data also demonstrated clear separation between patients and controls, confirming distinct metabolic profiles between the groups. Conclusions: Kynurenine pathway metabolites and enzymes show strong potential as non-invasive biomarkers for pituitary adenomas. In particular, elevated KAT and KYNA levels demonstrated high diagnostic performance. These findings suggest that a panel of kynurenine pathway metabolites could aid in the early, non-invasive detection of pituitary adenomas. Full article

The cDNA sequences of the common gonadotrophic hormone α-subunit (ag-Gthα) and of the specific follicle-stimulating hormone β-subunit (ag-Fshβ) of the giant Amazonian fish Arapaima gigas have been previously isolated by our research group. A synthesis of ag-Fsh in HEK293 cells and its purification and preliminary characterization were also carried out. In the present work, ag-Fsh was obtained and purified from the same host cells, and an extensive physical chemical characterization was performed via RP-HPLC, HPSEC, and MALDI-TOF-MS. Ag-Fsh, when compared to human FSH (hFSH), showed a higher hydrophobicity by RP-HPLC and a higher molecular mass (MM) via HPSEC. The same higher MM was also confirmed via MALDI-TOF-MS: 35,353 Da for ag-FSH against 31,969 Da for hFSH. Its immunological activity was also confirmed via an hFSH ELISA, in comparison with the highly purified pituitary preparation of hFSH-AFP7298A, from the National Hormone and Pituitary Program (NHPP-USA), with a clear response that was, however, 1560-fold lower when compared to the purest preparation. Finally, an in vitro bioassay, based on the stimulated release of 11-ketotestosterone (11-KT) from immature A. gigas testis, quantified ag-Fsh biological activity in comparison with human chorionic gonadotropin (hCG) and with human pituitary FSH-AFP7298A, showing a potency clearly higher than that of hCG. This suggests that injections of ag-Fsh in A. gigas and ag-Fsh cDNA gene therapy applications could be used for improving the reproductive functions of this threatened species. Full article

Background: Rabies virus (RABV) causes approximately 59,000 human deaths annually. Current pre- and post-exposure vaccination relies on inactivated vaccines (INVs) with limited yield and immunogenicity. We engineered a dual-cationic LNP-based nucleocapsid-like nanostructure (NLS) that co-encapsulates RABV G-mRNA and recombinant RABV-N to engage MHC-I/II pathways and enhance protection. Methods: A pVAX-RABV-G plasmid containing 5′/3′UTRs, Kozak, and poly(A) was transcribed in vitro. RABV-N with an N-terminal 6× His tag was expressed in E. coli BL21(DE3) and purified by Ni-Sepharose affinity chromatography. Dual-cationic LNPs (DHA, DOTAP Cl, mPEG-DTA2K, DOPC) were formulated by microfluidics at a 4:1 (G-mRNA:RABV-N) mass ratio. Vaccine quality was assessed by encapsulation efficiency, DLS, PDI, zeta potential, and TEM. Mice received empty LNPs, INV, G-mRNA, or NLS under varied schedules and doses. ELISA measured RABV-G/N-IgG; RFFIT determined neutralizing antibody (nAb) titers; ELISPOT quantified CTL response; qPCR assessed T-cell activation genes. On day 35 after the first immunization of vaccines, mice were challenged intramuscularly with 25 LD₅₀ of CVS-24. Results: G-mRNA purity was >95% and drove strong RABV-G expression in 293T cells. Purified RABV-N was approximately 52 kDa, >90% pure, and reactive to anti-His and anti-N antibodies. NLS achieved >95% encapsulation, a diameter of 136.9 nm, PDI 0.09, and a +18.7 mV zeta potential. A single dose yielded approximately 10 IU mL⁻¹ nAb by day 7; two doses peaked at approximately 1000 IU mL⁻¹. Mice showed 100% survival and no viral rebound in brain, spinal cord, and sciatic nerve. NLS induced stronger MHC-I/II-linked cellular immunity and higher RABV G/N-specific IFN-γ spot frequencies than G-mRNA or INV. Conclusions: The dual-antigen NLS vaccine co-delivering G-mRNA and RABV-N via dual-cationic LNPs robustly activates MHC-I/II, rapidly generates high-titer nAb (≥10 IU mL⁻¹ within 1 week), and sustains potent CD8⁺ CTL and CD4⁺ Th responses. A two-dose regimen (days 0 and 21) conferred complete protection, supporting the NLS platform as a next-generation rabies vaccine candidate. Full article

Catecholamines are fundamental mediators of the stress response, regulating arousal, vigilance, and adaptive behavior. However, their dynamics under extreme real-life conditions remain insufficiently explored. Survival, Evasion, Resistance, and Escape (SERE) training provides a unique model for examining neuroendocrine mechanisms of adaptation during both the acute phase and the recovery period following intense psychological and physical stress. Serum norepinephrine (NE) and dopamine (DA) were measured in 47 special forces soldiers during peak stress in SERE and one month later, compared with 17 healthy controls. Samples were collected under standardized conditions and analyzed using validated ELISA kits. NE levels differed significantly among groups (p = 0.003), being higher during SERE training and in controls compared to the post-recovery condition. DA also showed a significant group effect (p < 0.001), with increased levels during recovery and in soldiers during SERE relative to controls. The post-recovery decline in norepinephrine suggests adaptive habituation of sympathetic activity following extreme stress exposure. Conversely, the sustained elevation of dopamine during recovery may reflect neuroadaptive mechanisms that promote motivational and cognitive restoration. Together, these findings indicate coordinated catecholaminergic regulation supporting long-term resilience in elite military personnel. Full article

Sugarcane streak mosaic virus (SCSMV) is one of the mosaic viruses found in mixed infection with two or more viruses. Infections of mosaic viruses show highly similar mosaic symptoms and are difficult to distinguish. This study aimed to develop a specific antibody against SCSMV that could potentially be utilized to differentiate mosaic virus infections. The cDNA encoding the coat protein (CP) of SCSMV was isolated by RT-PCR from the symptomatic sugarcane leaves. The cDNA was then used for the production of CP for the development of its polyclonal antibody. The nucleotide sequence of the cDNA showed high homology of 94.2–97.3% at the amino acid level with the CP-cDNA of SCSMV isolated from India (AM749403.1), Africa (OR195142.1), and USA (U75456.1). CP was produced as a recombinant protein with a molecular size of 36.5 kDa in Escherichia coli. The injection of recombinant CP into a rabbit resulted in the production of polyclonal antibodies, which were used for the immunodetection of SCSMV in sugarcane. Immunoblot analysis revealed a specific reaction of SCSMV-CP in symptomatic sugarcane leaves. ELISA (Enzyme-Linked Immunosorbent Assay) and IC-RT-PCR (Immunocapture-Reverse Transcription-Polymerase Chain Reaction) using the CP antibody proved successful for detecting SCSMV infection in sugarcane leaves. The results indicate that the SCSMV-CP antibody is suitable for an immunodetection system and exhibits high specificity for SCSMV infection. Full article

Alcohol-associated liver disease is a global health burden with high morbidity and mortality, and the fungal microbiome is important for its progression. In particular, Candida albicans and C. albicans-reactive T helper 17 (Th17) cells contribute to alcohol-associated liver disease. Specific C. albicans antigens that activate Th17 cells during this disease are unknown. The C. albicans 65 kDa mannoprotein (CaMp65p) is one of the most abundant and immunodominant proteins of C. albicans, and is capable of eliciting robust T cell and interleukin (IL)-17A responses. The aim of this study was to measure levels of CaMp65p in serum of patients with alcohol use disorder and liver disease. Serum CaMp65p levels were measured in the serum of 60 patients with alcohol use disorder using an indirect competitive Enzyme-Linked Immunoabsorbent Assay (ELISA). Serum CaMp65p levels were correlated with liver disease severity. Serum CaMp65p levels positively correlated with several clinical and biochemical markers of liver injury and disease within the patient group with alcohol use disorder, including serum aspartate aminotransferase (AST; R = 0.33, p = 0.0092), alanine aminotransferase (ALT; R = 0.27, p = 0.037), gamma-glutamyl transferase (GGT; R = 0.35, p = 0.0055) and alkaline phosphatase (R = 0.36, p = 0.0052), and with the circulating M65 fragment of cytokeratin 18 (CK18-M65; R = 0.51, p = 0.0012), a marker of hepatocyte death. In addition, patients with alcohol use disorder in the upper quartile had significantly higher liver stiffness (p = 0.0022). Serum CaMp65p was significantly higher in patients with fibrosis stage F2–F4 as compared with patients with no or minimal fibrosis F0–F1 (p = 0.0082). The area under the curve (AUC) for detecting F2–F4 fibrosis was 0.70. Elevated serum CaMp65p levels are associated not only with more severe hepatic injury, but also with liver fibrosis in patients with alcohol use disorder. Therefore, CaMp65p may serve as a non-invasive biomarker for fibrosis assessment in patients with alcohol use disorder. Full article

Infectious hematopoietic necrosis virus (IHNV), a salmonid rhabdovirus, causes severe mortality exceeding 90% in both wild and farmed salmon and trout. Frequent outbreaks of IHNV highlight the urgent need for rapid detection methods to support effective prevention and control. This study developed a double-antibody sandwich ELISA (DAS-ELISA) targeting the nucleocapsid (N) protein of IHNV. Two peptides derived from the N protein—selected for their strong antigenicity, high level of conservation, and surface accessibility—were used as immunogens to generate two specific monoclonal antibodies. Following optimization, the DAS-ELISA was established using monoclonal antibody N-15 as the capture antibody and horseradish peroxidase (HRP)-conjugated antibody N-106 as the detection antibody. The results of this study demonstrated that DAS-ELISA exhibited high specificity for multiple IHNV strains and showed no cross-reactivity with IPNV, SVCV, or VHSV. The detection sensitivity of DAS-ELISA for IHNV was determined to be 10³ TCID₅₀/mL. Parallel analysis of 293 clinical samples using DAS-ELISA and WOAH reference method demonstrated a concordance rate of 92.83% (κ = 0.856). These results confirm that the established DAS-ELISA exhibits high sensitivity, specificity, broad-spectrum applicability, and repeatability. In conclusion, this DAS-ELISA provides a reliable and efficient tool for high-throughput early detection of IHNV infection in clinical settings. Full article

Background: The frequency of necrotising cutaneous and soft tissue infections caused by the Mucorales fungi Apophysomyces and Sakasenaea is increasing. The absence of sophisticated diagnostic technologies in low- and middle-income countries (LMICs) means that detection of cutaneous mucormycosis continues to rely on culture of the infecting pathogens from biopsy and their differentiation based on morphological characteristics. However, Apophysomyces and Sakasenaea are notorious for their failure to sporulate on standard mycological media used for the identification of human pathogenic fungi. Differentiation of these pathogens and their discrimination from Aspergillus fumigatus, the most common mould pathogen of humans, is essential due to their differing sensitivities to the antifungal drugs used to treat mucormycosis. Methods: A murine IgG1 monoclonal antibody, JD4, has been developed that is specific to Apophysomyces species. In Western blotting and enzyme-linked immunosorbent assay (ELISA), mAb JD4 is shown to bind to an extracellular 15 kDa protein, readily detectable in crude antigen extracts from non-sporulating cultures of Apophysomyces. Results: When combined with a Mucorales-specific lateral-flow immunoassay (LFIA), mAb JD4 allows the differentiation of Apophysomyces from Saksenaea species and discrimination from Aspergillus fumigatus. Monoclonal antibody JD4 enables the detection and differentiation of Apophysomyces species from other fungal pathogens that cause rapidly progressive cutaneous and soft tissue mycoses in humans. When this is combined with a rapid LFIA, improvements are offered in the sensitivity and specificity of Mucorales detection based on mycological culture, which remains a gold-standard procedure for mucormycosis detection in LMICs lacking access to more sophisticated diagnostic procedures. Full article

Porcine circovirus type 3 (PCV3), initially identified in the United States in 2016, is associated with multisystemic inflammation, myocarditis, reproductive failure in sows, and growth retardation in piglets, posing a significant economic threat to the swine industry. In this study, prokaryotic-expressed recombinant PCV3 Cap protein was used to immunize mice and rabbits. A monoclonal antibody (mAb 4G1) was generated through hybridoma technology, targeting a novel linear epitope (³⁷DYYDKK⁴²) within the first β-sheet of the Cap structure. This epitope exhibits high conservation (99.35%, 1239/1247) based on sequence alignment analysis, and residues 39 and 42 are critical residues affecting mAb binding. Subsequently, using rabbit polyclonal antibody (pAb) as the capture antibody and mAb 4G1 as the detection antibody, a double antibody sandwich ELISA (DAS-ELISA) method was developed. The assay demonstrates a cut-off value of 0.271, a detection limit for positive pig serum is 1:800, and shows no cross-reactivity with other swine pathogens. Intra- and inter-assay coefficients of variation were <10%, with a linear detection range for Cap protein down to 3.4 ng/mL. The coincidence rate between the DAS-ELISA and qPCR was 93.33% (70/75) for PCV3 detection in serum, with a kappa value of 0.837. This study establishes a simple, sensitive, and operationally efficient DAS-ELISA and provides a reference for monitoring PCV3 infection in swine herds. Full article

Staphylococcus aureus is a major pathogen responsible for staphylococcal food poisoning (SFP), with its pathogenicity primarily dependent on staphylococcal enterotoxins (SEs). Among these, staphylococcal enterotoxin A (SEA) is a critical risk factor due to its high toxicity, high detection rate (accounting for 80% of SFP cases), strong thermal stability, and resistance to hydrolysis. Traditional SEA immunoassays, such as enzyme-linked immunosorbent assay (ELISA), are prone to false-positive results caused by nonspecific binding interference from S. aureus surface protein A (SpA). In recent years, nanobodies (single-domain heavy-chain antibodies) have emerged as an ideal alternative to address SpA interference owing to their small molecular weight (15 kDa), high affinity, robust stability, and lack of Fc regions. In this study, based on a previously developed highly specific monoclonal antibody against SEA (mAb-4C6), four anti-SEA nanobodies paired with mAb-4C6 were obtained through two-part (four-round) of biopanning from a naive nanobody phage display library. Among these, SEA-4-20 and SEA-4-31 were selected as optimal candidates and paired with mAb-4C6 to construct double-antibody sandwich ELISAs. The detection limits for SEA were 0.135 ng/mL and 0.137 ng/mL, respectively, with effective elimination of SpA interference. This approach provides a reliable tool for rapid and accurate detection of SEA in food, clinical, and environmental samples. Full article

Sialyl-Tn (sTn) is a tumor-associated carbohydrate antigen (TACA) abundantly expressed by various types of carcinomas. While conventional antibody-based platforms have traditionally been used for the detection and targeting of sTn, alternative binding scaffolds may offer distinct advantages. Variable lymphocyte receptor B (VLRB), the immunoglobulin-like molecule of jawless vertebrates, offers a promising alternative for glycan recognition. In this study, a phage-displayed VLRB library was utilized to identify sTn-specific binders. Two candidates, designated as ccombodies A8 and B11, were isolated after four rounds of biopanning. Both were expressed and purified using Ni-affinity and FPLC, yielding proteins with apparent molecular weights of ~27 kDa in SDS-PAGE. Sequence analysis revealed a preference for glycan-binding residues in randomized hypervariable regions, with A8 exhibiting an increased aliphatic content. ELISA confirmed selective binding to sTn and other O-glycans containing the core α-GalNAc, with EC₅₀ values of 18.2 and 14.2 nM for A8 and B11, respectively. Vicia villosa lectin inhibited ccombody binding to sTn, indicating shared epitope recognition. Additionally, both ccombodies bound to sTn-positive glycoproteins and carcinoma cell lines HeLa and LS174T. These findings demonstrate that phage display of VLRBs enables the identification of high-affinity, glycan-specific binders, offering a compelling alternative to immunoglobulin-based platforms for future diagnostic and therapeutic applications targeting tumor-associated glycans. Full article

The objective of this study was to investigate the epidemiology of Fasciola hepatica in Cervus elaphus kept in natural conditions in the Riaño Regional Hunting Reserve, north-west of Spain, where several species of domestic and wild animals coexist. One hundred red deer were examined and classified according to age, sex, and sampling season. After the necropsy of the animals, the liver was removed and inspected to obtain the adult parasites of F. hepatica. Faecal samples were collected and processed using the coprological sedimentation technique. The prevalence of this trematode by necropsy was 12%, with a low number of specimens per animal ($\overline{\mathrm{x}}$ = 2.7 ± 1.5; range 1–6). The young animals and the males had a higher prevalence than the adults and the females, finding statistically significant differences only according to the host age. Significant variations were also observed when considering the seasons of the year, with the highest number of infected animals in spring. The histopathological study revealed the presence of lesions compatible with a chronic fasciolosis similar to that found in domestic animals. The shedding of F. hepatica eggs was quite low in terms of prevalence (6%) and mean intensity of infection ($\overline{\mathrm{x}}$ = 27.3 ± 20.6, range 4–60 epg), being in young animals, in males, and in spring, where the greatest excretion of eggs was observed. These results suggest that the deer are suitable definitive hosts for F. hepatica in the northwest of the Iberian Peninsula, but they are unusual hosts. The serum samples were analysed using a native excretory/secretory antigen (FhES) and a 2.9 kDa recombinant protein (FhrAPS) used for diagnosis of early and current fasciolosis in livestock. A commercial kit for serodiagnosis of F. hepatica in sheep and cattle, based on a monoclonal antibody (BIO K 211), was also evaluated in red deer. The seroprevalence of seropositivity of F. hepatica by FhES-ELISA was 32%, by FhrAPS-ELISA 13%, and by BIO K 211, 9%. In the three serological tests, the seroprevalence obtained was higher in adult animals, in males, and in spring. In the three serological tests used to understand the epidemiology of F. hepatica in red deer, we have observed that the sensitivity was low, perhaps due to the use of an anti-bovine IgG1 as a conjugate, so in future immunodiagnostic tests, it would be more desirable to obtain an anti-deer IgG, probably achieving better results. Due to these results, it is essential to investigate other serological or molecular tests that allow us to know the real importance of F. hepatica in deer and other wild animals. The role of deer as a reservoir of this trematode does not appear to be very important. Full article