Journal of Structural Biology

ScienceDirect RSS
  • The scrunchworm hypothesis: Transitions between A-DNA and B-DNA provide the driving force for genome packaging in double-stranded DNA bacteriophages
    [Jan 2015]

    Publication date: January 2015
    Source:Journal of Structural Biology, Volume 189, Issue 1

    Author(s): Stephen C. Harvey

    Double-stranded DNA bacteriophages have motors that drive the genome into preformed capsids, using the energy released by hydrolysis of ATP to overcome the forces opposing DNA packaging. Viral packaging motors are the strongest of all biological motors, but it is not known how they generate these forces. Several models for the process of mechanochemical force generation have been put forward, but there is no consensus on which, if any, of these is correct. All the existing models assume that protein-generated forces drive the DNA forward. The scrunchworm hypothesis proposes that the DNA molecule is the active force-generating core of the motor, not simply a substrate on which the motor operates. The protein components of the motor dehydrate a section of the DNA, converting it from the B form to the A form and shortening it by about 23%. The proteins then rehydrate the DNA, which converts back to the B form. Other regions of the motor grip and release the DNA to capture the shortening–lengthening motions of the B→A→B cycle (“scrunching”), so that DNA is pulled into the motor and pushed forward into the capsid. This DNA-centric mechanism provides a quantitative physical explanation for the magnitude of the forces generated by viral packaging motors. It also provides a simple explanation for the fact that each of the steps in the burst cycle advances the DNA by 2.5 base pairs. The scrunchworm hypothesis is consistent with a large body of published data, and it makes four experimentally testable predictions.





    Categories: Journal Articles
  • Improvement of protein binding sites prediction by selecting amino acid residues’ features
    [Jan 2015]

    Publication date: January 2015
    Source:Journal of Structural Biology, Volume 189, Issue 1

    Author(s): Georgina Mirceva , Andrea Kulakov

    One of the main focuses of bioinformatics community is the study of the relationship between the structure of the protein molecules and their functions. In the literature, there are various methods that consider different protein-derived information for predicting protein functions. In our research, we focus on predicting the protein binding sites, which could be used to functionally annotate the protein structures. In this paper we consider a set of sixteen amino acid residues’ features, and by applying various feature selection techniques we estimate their significance. Although the number of features in our case is not high, we perform feature selection in order to improve the prediction power and time complexity of the prediction models. The results show that by applying proper feature selection technique, the predictive performance of the classification algorithms is improved, i.e., by considering the most relevant features we induce more accurate models than if we consider the entire set of features. Furthermore, the model complexity, as well as the training and testing times are decreased by performing feature selection. We also compare our approach with several existing methods for protein binding sites prediction. The results demonstrate that the existing methods considered in this research are specific and applicable to the group of proteins for which the model was developed, while our approach is more generic and can be applied to a wider class of proteins.





    Categories: Journal Articles
  • Stability of the βB2B3 crystallin heterodimer to increased oxidation by radical probe and ion mobility mass spectrometry
    [Jan 2015]

    Publication date: January 2015
    Source:Journal of Structural Biology, Volume 189, Issue 1

    Author(s): Satoko Akashi , Simin D. Maleknia , Kazumi Saikusa , Kevin M. Downard

    Ion mobility mass spectrometry was employed to study the structure of the βB2B3-crystallin heterodimer following oxidation through its increased exposure to hydroxyl radicals. The results demonstrate that the heterodimer can withstand limited oxidation through the incorporation of up to some 10 oxygen atoms per subunit protein without any appreciable change to its average collision cross section and thus conformation. These results are in accord with the oxidation levels and timescales applicable to radical probe mass spectrometry (RP-MS) based protein footprinting experiments. Following prolonged exposure, the heterodimer is increasingly degraded through cleavage of the backbone of the subunit crystallins rather than denaturation such that heterodimeric structures with altered conformations and ion mobilities were not detected. However, evidence from measurements of oxidation levels within peptide segments, suggest the presence of some aggregated structure involving C-terminal domain segments of βB3 crystallin across residues 115–126 and 152–166. The results demonstrate, for the first time, the ability of ion mobility in conjunction with RP-MS to investigate the stability of protein complexes to, and the onset of, free radical based oxidative damage that has important implications in cataractogenesis.





    Categories: Journal Articles
  • Layered growth of crayfish gastrolith: About the stability of amorphous calcium carbonate and role of additives
    [Jan 2015]

    Publication date: January 2015
    Source:Journal of Structural Biology, Volume 189, Issue 1

    Author(s): Wouter J.E.M. Habraken , Admir Masic , Luca Bertinetti , Ali Al-Sawalmih , Lilah Glazer , Shmuel Bentov , Peter Fratzl , Amir Sagi , Barbara Aichmayer , Amir Berman

    Previous studies on pre-molt gastroliths have shown a typical onion-like morphology of layers of amorphous mineral (mostly calcium carbonate) and chitin, resulting from the continuous deposition and densification of amorphous mineral spheres on a chitin-matrix during time. To investigate the consequences of this layered growth on the local structure and composition of the gastrolith, we performed spatially-resolved Raman, X-ray and SEM–EDS analysis on complete pre-molt gastrolith cross-sections. Results show that especially the abundance of inorganic phosphate, phosphoenolpyruvate (PEP)/citrate and proteins is not uniform throughout the organ but changes from layer to layer. Based on these results we can conclude that ACC stabilization in the gastrolith takes place by more than one compound and not by only one of these additives.





    Categories: Journal Articles
  • Internal skeletal analysis of the colonial azooxanthellate scleractinian Dendrophyllia cribrosa using microfocus X-ray CT images: Underlying basis for its rigid and highly adaptive colony structure
    [Jan 2015]

    Publication date: January 2015
    Source:Journal of Structural Biology, Volume 189, Issue 1

    Author(s): Asuka Sentoku , Hitomi Morisaki , Shinji Masumoto , Rie Ohno , Takayuki Tomiyama , Yoichi Ezaki

    Dendrophyllid Scleractinia exhibit a variety of colonial morphologies, formed under the strict constraints on (1) budding sites, (2) orientations of the directive septa of offsets, (3) inclination of budding direction, and (4) those constraints in every generation. Dendrophyllia cribrosa exhibits a sympodial dendroid form, characteristically large coralla, and occasional fusions of adjacent branches within the same colony. Adjacent corallites are bound and supported by coenosteum skeleton. This study examined the inner skeletal structures at the junctions of fused branches using a non-destructive microfocus X-ray computed tomography (CT) imaging approach, and considered the reasons for the large colonial sizes and their adaptive significance. Three-dimensional reconstructions of two-dimensional X-ray CT images reveal that individual corallites are not directly connected in fused parts. Additionally, no completely buried individuals were found within fused skeleton. When adjacent branches approach one another, constituent corallites change their growth directions to avoid collisions between the branches. The adjacent branches fuse without a reduction in the number of constituent corallites, leading to the establishment of reticular and rigid colonial structures. In addition, a nearly even distribution of individuals on the colony surface facilitates efficient intake of nutrients. Thus, the growth of large D. cribrosa colonies involves avoidance of collision between constituent individuals, the reinforcement of colonial structure, and efficient uptake of nutrients. These observations provide insights on the dynamics of interrelationships between colony-making mechanisms and the adaptive strategies required under habitat conditions such as specific current activities.





    Categories: Journal Articles
  • Quantitative analysis of mouse pancreatic islet architecture by serial block-face SEM
    [Jan 2015]

    Publication date: January 2015
    Source:Journal of Structural Biology, Volume 189, Issue 1

    Author(s): C.R. Pfeifer , A. Shomorony , M.A. Aronova , G. Zhang , T. Cai , H. Xu , A.L. Notkins , R.D. Leapman

    We have applied serial block-face scanning electron microscopy (SBF-SEM) to measure parameters that describe the architecture of pancreatic islets of Langerhans, microscopic endocrine organs that secrete insulin and glucagon for control of blood glucose. By analyzing entire mouse islets, we show that it is possible to determine (1) the distributions of alpha and beta cells, (2) the organization of blood vessels and pericapillary spaces, and (3) the ultrastructure of the individual secretory cells. Our results show that the average volume of a beta cell is nearly twice that of an alpha cell, and the total mitochondrial volume is about four times larger. In contrast, nuclear volumes in the two cell types are found to be approximately equal. Although the cores of alpha and beta secretory granules have similar diameters, the beta granules have prominent halos resulting in overall diameters that are twice those of alpha granules. Visualization of the blood vessels revealed that every secretory cell in the islet is in contact with the pericapillary space, with an average contact area of 9±5% of the cell surface area. Our data show that consistent results can be obtained by analyzing small numbers of islets. Due to the complicated architecture of pancreatic islets, such precision cannot easily be achieved by using TEM of thin sections.





    Categories: Journal Articles
  • Analysis of acute brain slices by electron microscopy: A correlative light–electron microscopy workflow based on Tokuyasu cryo-sectioning
    [Jan 2015]

    Publication date: January 2015
    Source:Journal of Structural Biology, Volume 189, Issue 1

    Author(s): Celine Loussert Fonta , Andrew Leis , Cliff Mathisen , David S. Bouvier , Willy Blanchard , Andrea Volterra , Ben Lich , Bruno M. Humbel

    Acute brain slices are slices of brain tissue that are kept vital in vitro for further recordings and analyses. This tool is of major importance in neurobiology and allows the study of brain cells such as microglia, astrocytes, neurons and their inter/intracellular communications via ion channels or transporters. In combination with light/fluorescence microscopies, acute brain slices enable the ex vivo analysis of specific cells or groups of cells inside the slice, e.g. astrocytes. To bridge ex vivo knowledge of a cell with its ultrastructure, we developed a correlative microscopy approach for acute brain slices. The workflow begins with sampling of the tissue and precise trimming of a region of interest, which contains GFP-tagged astrocytes that can be visualised by fluorescence microscopy of ultrathin sections. The astrocytes and their surroundings are then analysed by high resolution scanning transmission electron microscopy (STEM). An important aspect of this workflow is the modification of a commercial cryo-ultramicrotome to observe the fluorescent GFP signal during the trimming process. It ensured that sections contained at least one GFP astrocyte. After cryo-sectioning, a map of the GFP-expressing astrocytes is established and transferred to correlation software installed on a focused ion beam scanning electron microscope equipped with a STEM detector. Next, the areas displaying fluorescence are selected for high resolution STEM imaging. An overview area (e.g. a whole mesh of the grid) is imaged with an automated tiling and stitching process. In the final stitched image, the local organisation of the brain tissue can be surveyed or areas of interest can be magnified to observe fine details, e.g. vesicles or gold labels on specific proteins. The robustness of this workflow is contingent on the quality of sample preparation, based on Tokuyasu’s protocol. This method results in a reasonable compromise between preservation of morphology and maintenance of antigenicity. Finally, an important feature of this approach is that the fluorescence of the GFP signal is preserved throughout the entire preparation process until the last step before electron microscopy.





    Categories: Journal Articles
  • The structure of ibuprofen bound to cyclooxygenase-2
    [Jan 2015]

    Publication date: January 2015
    Source:Journal of Structural Biology, Volume 189, Issue 1

    Author(s): Benjamin J. Orlando , Michael J. Lucido , Michael G. Malkowski

    The cyclooxygenases (COX-1 and COX-2) catalyze the rate-limiting step in the biosynthesis of prostaglandins, and are the pharmacological targets of non-steroidal anti-inflammatory drugs (NSAIDs) and COX-2 selective inhibitors (coxibs). Ibuprofen (IBP) is one of the most commonly available over-the-counter pharmaceuticals in the world. The anti-inflammatory and analgesic properties of IBP are thought to arise from inhibition of COX-2 rather than COX-1. While an X-ray crystal structure of IBP bound to COX-1 has been solved, no such structure exists for the cognate isoform COX-2. We have determined the crystal structure of muCOX-2 with a racemic mixture of (R/S)-IBP. Our structure reveals that only the S-isomer of IBP was bound, indicating that the S-isomer possesses higher affinity for COX-2 than the R-isomer. Mutational analysis of Arg-120 and Tyr-355 at the entrance of the cyclooxygenase channel confirmed their role in binding and inhibition of COX-2 by IBP. Our results provide the first atomic level detail of the interaction between IBP and COX-2.





    Categories: Journal Articles
  • Structure of the c-Src-SH3 domain in complex with a proline-rich motif of NS5A protein from the hepatitis C virus
    [Jan 2015]

    Publication date: January 2015
    Source:Journal of Structural Biology, Volume 189, Issue 1

    Author(s): Julio Bacarizo , Sergio Martínez-Rodríguez , Ana Cámara-Artigas

    The non-structural hepatitis C virus proteins NS5A and NS5B form a complex through interaction with the SH2 and SH3 domains of the non-receptor Src tyrosine kinase, which seems essential for viral replication. We have crystallized the complex between the SH3 domain of the c-Src tyrosine kinase and the C-terminal proline rich motif of the NS5A protein (A349PPIPPPRRKR359). Crystals obtained at neutral pH belong to the space group I41, with a single molecule of the SH3/NS5A complex at the asymmetric unit. The NS5A peptide is bound in a reverse orientation (class II) and the comparison of this structure with those of the high affinity synthetic peptides APP12 and VSL12 shows some important differences at the salt bridge that drives the peptide orientation. Further conformational changes in residues placed apart from the binding site also seem to play an important role in the binding orientation of this peptide. Our results show the interaction of the SH3 domain of the c-Src tyrosine kinase with a proline rich motif in the NS5A protein and point to their potential interaction in vivo.





    Categories: Journal Articles
  • Large area sub-micron chemical imaging of magnesium in sea urchin teeth
    [Jan 2015]

    Publication date: Available online 31 December 2014
    Source:Journal of Structural Biology

    Author(s): Admir Masic , James C. Weaver

    The heterogeneous and site-specific incorporation of inorganic ions can profoundly influence the local mechanical properties of damage tolerant biological composites. Using the sea urchin tooth as a research model, we describe a multi-technique approach to spatially map the distribution of magnesium in this complex multiphase system. Through the combined use of 16-bit backscattered scanning electron microscopy, multi-channel energy dispersive spectroscopy elemental mapping, and diffraction-limited confocal Raman spectroscopy, we demonstrate a new set of high throughput, multi-spectral, high resolution methods for the large scale characterization of mineralized biological materials. In addition, instrument hardware and data collection protocols can be modified such that several of these measurements can be performed on irregularly shaped samples with complex surface geometries and without the need for extensive sample preparation. Using these approaches, in conjunction with whole animal micro-computed tomography studies, we have been able to spatially resolve micron and sub-micron structural features across macroscopic length scales on entire urchin tooth cross-sections and correlate these complex morphological features with local variability in elemental composition.





    Categories: Journal Articles
  • An N-terminal extension to the hepatitis B virus core protein forms a poorly ordered trimeric spike in assembled virus-like particles
    [Jan 2015]

    Publication date: Available online 31 December 2014
    Source:Journal of Structural Biology

    Author(s): Richard McGonigle , Wei Boon Yap , Swee Tin Ong , Derek Gatherer , Saskia E. Bakker , Wen Siang Tan , David Bhella

    Virus-like particles composed of the core antigen of hepatitis B virus (HBcAg) have been shown to be an effective platform for the display of foreign epitopes in vaccine development. Heterologous sequences have been successfully inserted at both amino and carboxy termini as well as internally at the major immunodominant epitope. We used cryogenic electron microscopy (CryoEM) and three-dimensional image reconstruction to investigate the structure of VLPs assembled from an N-terminal extended HBcAg that contained a polyhistidine tag. The insert was seen to form a trimeric spike on the capsid surface that was poorly resolved, most likely owing to it being flexible. We hypothesise that the capacity of N-terminal inserts to form trimers may have application in the development of multivalent vaccines to trimeric antigens. Our analysis also highlights the value of tools for local resolution assessment in studies of partially disordered macromolecular assemblies by cryoEM.





    Categories: Journal Articles
  • Use of a “silver bullet” to resolve crystal lattice dislocation disorder: A cobalamin complex of Δ1-pyrroline-5-carboxylate dehydrogenase from Mycobacterium tuberculosis
    [Jan 2015]

    Publication date: Available online 31 December 2014
    Source:Journal of Structural Biology

    Author(s): Thomas Lagautriere , Ghader Bashiri , Edward N. Baker

    The use of small molecules as “silver bullets” that can bind to generate crosslinks between protein molecules has been advanced as a powerful means of enhancing success in protein crystallization (McPherson and Cudney, 2006). We have explored this approach in attempts to overcome an order–disorder phenomenon that complicated the structural analysis of the enzyme Δ1-pyrroline-5-carboxylate dehydrogenase from Mycobacterium tuberculosis (P5CDH, Mtb-PruA). Using the Silver Bullets Bio screen, we obtained new crystal packing using cobalamin as a co-crystallization agent. This crystal form did not display the order–disorder phenomenon previously encountered. Solution of the crystal structure showed that cobalamin molecules are present in the crystal contacts. Although the cobalamin binding probably does not have physiological relevance, it reflects similarities in the nucleotide-binding region of Mtb-PruA, with the nucleotide loop of cobalamin sharing the binding site for the adenine moiety of NAD+.





    Categories: Journal Articles
  • Seeing tobacco mosaic virus through direct electron detectors
    [Jan 2015]

    Publication date: Available online 17 December 2014
    Source:Journal of Structural Biology

    Author(s): Simon A. Fromm , Tanmay A.M. Bharat , Arjen J. Jakobi , Wim J.H. Hagen , Carsten Sachse

    With the introduction of direct electron detectors (DED) to the field of electron cryo-microscopy, a wave of atomic-resolution structures has become available. As the new detectors still require comparative characterization, we have used tobacco mosaic virus (TMV) as a test specimen to study the quality of 3D image reconstructions from data recorded on the two direct electron detector cameras, K2 Summit and Falcon II. Using DED movie frames, we explored related image-processing aspects and compared the performance of micrograph-based and segment-based motion correction approaches. In addition, we investigated the effect of dose deposition on the atomic-resolution structure of TMV and show that radiation damage affects negative carboxyl chains first in a side-chain specific manner. Finally, using 450,000 asymmetric units and limiting the effects of radiation damage, we determined a high-resolution cryo-EM map at 3.35Å resolution. Here, we provide a comparative case study of highly ordered TMV recorded on different direct electron detectors to establish recording and processing conditions that enable structure determination up to 3.2Å in resolution using cryo-EM.





    Categories: Journal Articles
  • Tomo3D 2.0 – Exploitation of Advanced Vector eXtensions (AVX) for 3D reconstruction
    [Jan 2015]

    Publication date: Available online 17 December 2014
    Source:Journal of Structural Biology

    Author(s): Jose-Ignacio Agulleiro , Jose-Jesus Fernandez

    Tomo3D is a program for fast tomographic reconstruction on multicore computers. Its high speed stems from code optimization, vectorization with Streaming SIMD Extensions (SSE), multithreading and optimization of disk access. Recently, Advanced Vector eXtensions (AVX) have been introduced in the x86 processor architecture. Compared to SSE, AVX double the number of simultaneous operations, thus pointing to a potential twofold gain in speed. However, in practice, achieving this potential is extremely difficult. Here, we provide a technical description and an assessment of the optimizations included in Tomo3D to take advantage of AVX instructions. Tomo3D 2.0 allows huge reconstructions to be calculated in standard computers in a matter of minutes. Thus, it will be a valuable tool for electron tomography studies with increasing resolution needs.





    Categories: Journal Articles
  • On the use of Legionella/Rickettsia chimeras to investigate the structure and regulation of Rickettsia effector RalF
    [Jan 2015]

    Publication date: Available online 9 December 2014
    Source:Journal of Structural Biology

    Author(s): Marcia Folly-Klan , Bastien Sancerne , Eric Alix , Craig R. Roy , Jacqueline Cherfils , Valérie Campanacci

    A convenient strategy to interrogate the biology of regulatory proteins is to replace individual domains by an equivalent domain from a related protein of the same species or from an ortholog of another species. It is generally assumed that the overall properties of the native protein are retained in the chimera, and that functional differences reflect only the specific determinants contained in the swapped domains. Here we used this strategy to circumvent the difficulty in obtaining crystals of Rickettsia prowazekii RalF, a bacterial protein that functions as a guanine nucleotide exchange factor for eukaryotic Arf GTPases. A RalF homolog is encoded by Legionella pneumophila, in which a C-terminal capping domain auto-inhibits the catalytic Sec7 domain and localizes the protein to the Legionella-containing vacuole. The crystal structures of domain-swapped chimeras were determined and used to construct a model of Legionella RalF with a RMSD of less than 1Å with the crystal structure, which validated the use of this approach to build a model of Rickettsia RalF. In the Rickettsia RalF model, sequence differences in the capping domain that target it to specific membranes are accommodated by a shift of the entire domain with respect to the Sec7 domain. However, local sequence changes also give rise to an artifactual salt bridge in one of the chimeras, which likely explains why this chimera is recalcitrant to activation. These findings highlight the structural plasticity whereby chimeras can be engineered, but also underline that unpredictable differences can modify their biochemical responses.





    Categories: Journal Articles
  • Semi-automated selection of cryo-EM particles in RELION-1.3
    [Jan 2015]

    Publication date: Available online 6 December 2014
    Source:Journal of Structural Biology

    Author(s): Sjors H.W. Scheres

    The selection of particles suitable for high-resolution cryo-EM structure determination from noisy micrographs may represent a tedious and time-consuming step. Here, a semi-automated particle selection procedure is presented that has been implemented within the open-source software RELION. At the heart of the procedure lies a fully CTF-corrected template-based picking algorithm, which is supplemented by a fast sorting algorithm and reference-free 2D class averaging to remove false positives. With only limited user-interaction, the proposed procedure yields results that are comparable to manual particle selection. Together with an improved graphical user interface, these developments further contribute to turning RELION from a stand-alone refinement program into a convenient image processing pipeline for the entire single-particle approach.





    Categories: Journal Articles
  • Third Harmonic Generation microscopy as a reliable diagnostic tool for evaluating lipid body modification during cell activation: The example of BV-2 microglia cells
    [Jan 2015]

    Publication date: Available online 5 December 2014
    Source:Journal of Structural Biology

    Author(s): E. Gavgiotaki , G. Filippidis , M. Kalognomou , A.A. Tsouko , I. Skordos , C. Fotakis , I. Athanassakis

    Nonlinear optical processes have found widespread applications in fields ranging from fundamental physics to biomedicine. In this study, we attempted to evaluate cell activation by using the Third Harmonic Generation (THG) imaging microscopy as a new diagnostic tool. The BV-2 microglia cell line with or without activation by lipopolysaccharide was chosen as a representative biological model. The results showed that THG imaging could discriminate between the control versus activated state of BV-2 cells not only as to THG signal intensity but also as to THG signal area, while verifying once more that the majority of the intracellular detected signal corresponds to lipid bodies. Since THG imaging is a real time, non-destructive modality and does not require any prior cell processing and staining, the results presented here provide an important tool for normal versus activated cell discrimination, which could be proved very useful in the study of inflammation.





    Categories: Journal Articles
  • Sparse and incomplete factorial matrices to screen membrane protein 2D crystallization
    [Jan 2015]

    Publication date: Available online 3 December 2014
    Source:Journal of Structural Biology

    Author(s): R. Lasala , N. Coudray , A. Abdine , Z. Zhang , M. Lopez-Redondo , R. Kirshenbaum , J. Alexopoulos , Z. Zolnai , D.L. Stokes , I. Ubarretxena-Belandia

    Electron crystallography is well suited for studying the structure of membrane proteins in their native lipid bilayer environment. This technique relies on electron cryomicroscopy of two-dimensional (2D) crystals, grown generally by reconstitution of purified membrane proteins into proteoliposomes under conditions favoring the formation of well-ordered lattices. Growing these crystals presents one of the major hurdles in the application of this technique. To identify conditions favoring crystallization a wide range of factors that can lead to a vast matrix of possible reagent combinations must be screened. However, in 2D crystallization these factors have traditionally been surveyed in a relatively limited fashion. To address this problem we carried out a detailed analysis of published 2D crystallization conditions for 12 β-barrel and 138 α-helical membrane proteins. From this analysis we identified the most successful conditions and applied them in the design of new sparse and incomplete factorial matrices to screen membrane protein 2D crystallization. Using these matrices we have run 19 crystallization screens for 16 different membrane proteins totaling over 1300 individual crystallization conditions. Six membrane proteins have yielded diffracting 2D crystals suitable for structure determination, indicating that these new matrices show promise to accelerate the success rate of membrane protein 2D crystallization.





    Categories: Journal Articles
  • Cover 2 - Editorial Board
    [Jan 2015]

    Publication date: December 2014
    Source:Journal of Structural Biology, Volume 188, Issue 3









    Categories: Journal Articles
  • Table of Contents / barcode
    [Jan 2015]

    Publication date: December 2014
    Source:Journal of Structural Biology, Volume 188, Issue 3









    Categories: Journal Articles