Structure-Mechanics Principles and Mechanobiology of Fibrocartilage Pericellular Matrix: A Pivotal Role of Type V Collagen.

The pericellular matrix (PCM) is the immediate microniche surrounding resident cells in various tissue types, regulating matrix turnover, cell-matrix cross-talk and disease initiation. This study elucidated the structure-mechanical properties and mechanobiological functions of the PCM in fibrocartilage, a family of connective tissues that sustain complex tensile and compressive loads in vivo. Studying the murine meniscus as the model tissue, we showed that fibrocartilage PCM contains thinner, random collagen fibrillar networks that entrap proteoglycans, a structure distinct from the densely packed, highly aligned collagen fibers in the bulk extracellular matrix (ECM). In comparison to the ECM, the PCM has a lower modulus and greater isotropy, but similar relative viscoelastic properties. In Col5a1 +/- menisci, the reduction of collagen V, a minor collagen localized in the PCM, resulted in aberrant fibril thickening with increased heterogeneity. Consequently, the PCM exhibited a reduced modulus, loss of isotropy and faster viscoelastic relaxation. This disrupted PCM contributes to perturbed mechanotransduction of resident meniscal cells, as illustrated by reduced intracellular calcium signaling, as well as upregulated biosynthesis of lysyl oxidase and tenascin C. When cultured in vitro, Col5a1 +/- meniscal cells synthesized a weakened nascent PCM, which had inferior properties towards protecting resident cells against applied tensile stretch. These findings underscore the PCM as a distinctive microstructure that governs fibrocartilage mechanobiology, and highlight the pivotal role of collagen V in PCM function. Targeting the PCM or its molecular constituents holds promise for enhancing not only meniscus regeneration and osteoarthritis intervention, but also addressing diseases across various fibrocartilaginous tissues.

Low-Temperature Nanosecond Laser Process of HZO-IGZO FeFETs toward Monolithic 3D System on Chip Integration.

Ferroelectric field-effect transistors (FeFETs) are increasingly important for in-memory computing and monolithic 3D (M3D) integration in system-on-chip (SoC) applications. However, the high-temperature processing required by most ferroelectric memories can lead to thermal damage to the underlying device layers, which poses significant physical limitations for 3D integration processes. To solve this problem, the study proposes using a nanosecond pulsed laser for selective annealing of hafnia-based FeFETs, enabling precise control of heat penetration depth within thin films. Sufficient thermal energy is delivered to the IGZO oxide channel and HZO ferroelectric gate oxide without causing thermal damage to the bottom layer, which has a low transition temperature (<250 °C). Using optimized laser conditions, a fast response time (<1 µs) and excellent stability (cycle > 106, retention > 106 s) are achieved in the ferroelectric HZO film. The resulting FeFET exhibited a wide memory window (>1.7 V) with a high on/off ratio (>105). In addition, moderate ferroelectric properties (2·Pr of 14.7 µC cm-2) and pattern recognition rate-based linearity (potentiation: 1.13, depression: 1.6) are obtained. These results demonstrate compatibility in HZO FeFETs by specific laser annealing control and thin-film layer design for various structures (3D integrated, flexible) with neuromorphic applications.

Survivin as a mediator of stiffness-induced cell cycle progression and proliferation of vascular smooth muscle cells.

Stiffened arteries are a pathology of atherosclerosis, hypertension, and coronary artery disease and a key risk factor for cardiovascular disease events. The increased stiffness of arteries triggers a phenotypic switch, hypermigration, and hyperproliferation of vascular smooth muscle cells (VSMCs), leading to neointimal hyperplasia and accelerated neointima formation. However, the mechanism underlying this trigger remains unknown. Our analyses of whole-transcriptome microarray data from mouse VSMCs cultured on stiff hydrogels simulating arterial pathology identified 623 genes that were significantly and differentially expressed (360 upregulated and 263 downregulated) relative to expression in VSMCs cultured on soft hydrogels. Functional enrichment and gene network analyses revealed that these stiffness-sensitive genes are linked to cell cycle progression and proliferation. Importantly, we found that survivin, an inhibitor of apoptosis protein, mediates stiffness-dependent cell cycle progression and proliferation as determined by gene network and pathway analyses, RT-qPCR, immunoblotting, and cell proliferation assays. Furthermore, we found that inhibition of cell cycle progression did not reduce survivin expression, suggesting that survivin functions as an upstream regulator of cell cycle progression and proliferation in response to ECM stiffness. Mechanistically, we found that the stiffness signal is mechanotransduced via the FAK-E2F1 signaling axis to regulate survivin expression, establishing a regulatory pathway for how the stiffness of the cellular microenvironment affects VSMC behaviors. Overall, our findings indicate that survivin is necessary for VSMC cycling and proliferation and plays a role in regulating stiffness-responsive phenotypes.

Survivin regulates intracellular stiffness and extracellular matrix production in vascular smooth muscle cells.

Vascular dysfunction is a common cause of cardiovascular diseases characterized by the narrowing and stiffening of arteries, such as atherosclerosis, restenosis, and hypertension. Arterial narrowing results from the aberrant proliferation of vascular smooth muscle cells (VSMCs) and their increased synthesis and deposition of extracellular matrix (ECM) proteins. These, in turn, are modulated by arterial stiffness, but the mechanism for this is not fully understood. We found that survivin is an important regulator of stiffness-mediated ECM synthesis and intracellular stiffness in VSMCs. Whole-transcriptome analysis and cell culture experiments showed that survivin expression is upregulated in injured femoral arteries in mice and in human VSMCs cultured on stiff fibronectin-coated hydrogels. Suppressed expression of survivin in human VSMCs significantly decreased the stiffness-mediated expression of ECM components related to arterial stiffening, such as collagen-I, fibronectin, and lysyl oxidase. By contrast, expression of these ECM proteins was rescued by ectopic expression of survivin in human VSMCs cultured on soft hydrogels. Interestingly, atomic force microscopy analysis showed that suppressed or ectopic expression of survivin decreases or increases intracellular stiffness, respectively. Furthermore, we observed that inhibiting Rac and Rho reduces survivin expression, elucidating a mechanical pathway connecting intracellular tension, mediated by Rac and Rho, to survivin induction. Finally, we found that survivin inhibition decreases FAK phosphorylation, indicating that survivin-dependent intracellular tension feeds back to maintain signaling through FAK. These findings suggest a novel mechanism by which survivin potentially modulates arterial stiffness.

Vacuum Tunneling Transistor with Nano Vacuum Chamber for Harsh Environments.

A nano vacuum tube which consists of a vacuum transistor and a nano vacuum chamber was demonstrated. For the device, a vacuum region is an electron transport channel, and a vacuum is a tunneling barrier. Tilted angle evaporation was studied for the formation of the nano level vacuum chamber structure. This vacuum tube was ultraminiaturized with several tens of 10-18 L scale volume and 10-6 Torr of pressure. The device structure made it possible to achieve a high integration density and to sustain the vacuum state in various real operations. In particular, the vacuum transistor performed stably in extreme external environments because the tunneling mechanism showed a wide range of working stability. The vacuum was sustained well by the sealing layer and provided a defect-free tunneling junction. In tests, the high vacuum level was maintained for more than 15 months with high reliability. The Al sealing layer and tube structure can effectively block exposed light such as visible light and UV, enabling the stable operation of the tunneling transistor. In addition, it is estimated that the structure blocks approximately 5 keV of X-ray. The device showed stable operating characteristics in a wide temperature range of 100-390 K. Therefore, the vacuum tube can be used in a wide range of applications involving integrated circuits while resolving the disadvantages of a large volume in old vacuum tubes. Additionally, it can be an important solution for next-generation devices in various fields such as aerospace, artificial intelligence, and THz applications.

Double Digital Assay for Single Extracellular Vesicle and Single Molecule Detection.

Extracellular vesicles (EVs) have emerged as a promising source of biomarkers for disease diagnosis. However, current diagnostic methods for EVs present formidable challenges, given the low expression levels of biomarkers carried by EV samples, as well as their complex physical and biological properties. Herein, a highly sensitive double digital assay is developed that allows for the absolute quantification of individual molecules from a single EV. Because the relative abundance of proteins is low for a single EV, tyramide signal amplification (TSA) is integrated to increase the fluorescent signal readout for evaluation. With the integrative microfluidic technology, the technology's ability to compartmentalize single EVs is successfully demonstrated, proving the technology's digital partitioning capacity. Then the device is applied to detect single PD-L1 proteins from single EVs derived from a melanoma cell line and it is discovered that there are ≈2.7 molecules expressed per EV, demonstrating the applicability of the system for profiling important prognostic and diagnostic cancer biomarkers for therapy response, metastatic status, and tumor progression. The ability to accurately quantify protein molecules of rare abundance from individual EVs will shed light on the understanding of EV heterogeneity and discovery of EV subtypes as new biomarkers.

Suspension bath bioprinting and maturation of anisotropic meniscal constructs.

Due to limited intrinsic healing capacity of the meniscus, meniscal injuries pose a significant clinical challenge. The most common method for treatment of damaged meniscal tissues, meniscectomy, leads to improper loading within the knee joint, which can increase the risk of osteoarthritis. Thus, there is a clinical need for the development of constructs for meniscal repair that better replicate meniscal tissue organization to improve load distributions and function over time. Advanced three-dimensional bioprinting technologies such as suspension bath bioprinting provide some key advantages, such as the ability to support the fabrication of complex structures using non-viscous bioinks. In this work, the suspension bath printing process is utilized to print anisotropic constructs with a unique bioink that contains embedded hydrogel fibers that align via shear stresses during printing. Constructs with and without fibers are printed and then cultured for up to 56 din vitroin a custom clamping system. Printed constructs with fibers demonstrate increased cell and collagen alignment, as well as enhanced tensile moduli when compared to constructs printed without fibers. This work advances the use of biofabrication to develop anisotropic constructs that can be utilized for the repair of meniscal tissue.

Aberrant chromatin reorganization in cells from diseased fibrous connective tissue in response to altered chemomechanical cues.

Changes in the micro-environment of fibrous connective tissue can lead to alterations in the phenotypes of tissue-resident cells, yet the underlying mechanisms are poorly understood. Here, by visualizing the dynamics of histone spatial reorganization in tenocytes and mesenchymal stromal cells from fibrous tissue of human donors via super-resolution microscopy, we show that physiological and pathological chemomechanical cues can directly regulate the spatial nanoscale organization and density of chromatin in these tissue-resident cell populations. Specifically, changes in substrate stiffness, altered oxygen tension and the presence of inflammatory signals drive chromatin relocalization and compaction into the nuclear boundary, mediated by the activity of the histone methyltransferase EZH2 and an intact cytoskeleton. In healthy cells, chemomechanically triggered changes in the spatial organization and density of chromatin are reversible and can be attenuated by dynamically stiffening the substrate. In diseased human cells, however, the link between mechanical or chemical inputs and chromatin remodelling is abrogated. Our findings suggest that aberrant chromatin organization in fibrous connective tissue may be a hallmark of disease progression that could be leveraged for therapeutic intervention.

A machine learning pipeline revealing heterogeneous responses to drug perturbations on vascular smooth muscle cell spheroid morphology and formation.

Machine learning approaches have shown great promise in biology and medicine discovering hidden information to further understand complex biological and pathological processes. In this study, we developed a deep learning-based machine learning algorithm to meaningfully process image data and facilitate studies in vascular biology and pathology. Vascular injury and atherosclerosis are characterized by neointima formation caused by the aberrant accumulation and proliferation of vascular smooth muscle cells (VSMCs) within the vessel wall. Understanding how to control VSMC behaviors would promote the development of therapeutic targets to treat vascular diseases. However, the response to drug treatments among VSMCs with the same diseased vascular condition is often heterogeneous. Here, to identify the heterogeneous responses of drug treatments, we created an in vitro experimental model system using VSMC spheroids and developed a machine learning-based computational method called HETEROID (heterogeneous spheroid). First, we established a VSMC spheroid model that mimics neointima-like formation and the structure of arteries. Then, to identify the morphological subpopulations of drug-treated VSMC spheroids, we used a machine learning framework that combines deep learning-based spheroid segmentation and morphological clustering analysis. Our machine learning approach successfully showed that FAK, Rac, Rho, and Cdc42 inhibitors differentially affect spheroid morphology, suggesting that multiple drug responses of VSMC spheroid formation exist. Overall, our HETEROID pipeline enables detailed quantitative drug characterization of morphological changes in neointima formation, that occurs in vivo, by single-spheroid analysis.

Miniaturized Self-Resonant Micro Coil Array with A Floating Structure for Wireless Multi-Channel Transmission.

Micro size antennas have significant merits due to the small size effect, enabling new device concepts. However, the low-quality factor (Q-factor), the large size of impedance matching components, and the poor selectivity of the multi-array design remain challenging issues. To solve these issues, a floating coil structure stacked on a loop micro-antenna is suggested. Various floating coil designs are prepared with appropriate matching conditions at specific target frequencies, using an easy fabrication process without the need for additional space. A simple one-loop antenna design shows a higher Q-factor than other, more complicated designs. The micro-sized loop antenna with the 80 µm trace width design exhibits the highest Q-factor, around 31 within 7 GHz. The 8 different floating coil designs result in high-frequency selectivity from 1 to 7 GHz. The highest selectivity contrast and WPT efficiency are above 7 and around 1%, respectively. Considering the size of the antenna, the efficiency is not low, mainly due to the good matching effect with the high Q-factor of the floating coil and the loop antenna. This micro-antenna array concept with high integration density can be applied for advanced wireless neural stimulation or in wireless pixel array concepts in flexible displays.

Stretch-responsive adhesive microcapsules for strain-regulated antibiotic release from fabric wound dressings.

Bacterial infection of a wound is a major complication that can significantly delay proper healing and even necessitate surgical debridement. Conventional non-woven fabric dressings, including gauzes, bandages and cotton wools, often fail in treating wound infections in a timely manner due to their passive release mechanism of antibiotics. Here, we propose adhesive mechanically-activated microcapsules (MAMCs) capable of strongly adhering to a fibrous matrix to achieve a self-regulated release of antibiotics upon uniaxial stretching of non-woven fabric dressings. To achieve this, a uniform population of polydopamine (PDA)-coated MAMCs (PDA-MAMCs) are prepared using a microfluidics technique and subsequent oxidative dopamine polymerization. The PDA-MAMC allows for robust mechano-activation within the fibrous network through high retention and effective transmission of mechanical force under stretching. By validating the potential of a PDA-MAMCs-laden gauze to release antibiotics in a tensile strain-dependent manner, we demonstrate that PDA-MAMCs can be successfully incorporated into a woven material and create a smart wound dressing for control of bacterial infections. This new mechano-activatable delivery approach will open up a new avenue for a stretch-triggered, on-demand release of therapeutic cargos in skin-mountable or wearable biomedical devices.

3D cell aggregate printing technology and its applications.

Various cell aggregate culture technologies have been developed and actively applied to tissue engineering and organ-on-a-chip. However, the conventional culture technologies are labor-intensive, and their outcomes are highly user dependent. In addition, the technologies cannot be used to produce three-dimensional (3D) complex tissues. In this regard, 3D cell aggregate printing technology has attracted increased attention from many researchers owing to its 3D processability. The technology allows the fabrication of 3D freeform constructs using multiple types of cell aggregates in an automated manner. Technological advancement has resulted in the development of a printing technology with a high resolution of approximately 20 µm in 3D space. A high-speed printing technology that can print a cell aggregate in milliseconds has also been introduced. The developed aggregate printing technologies are being actively applied to produce various types of engineered tissues. Although various types of high-performance printing technologies have been developed, there are still some technical obstacles in the fabrication of engineered tissues that mimic the structure and function of native tissues. This review highlights the central importance and current technical level of 3D cell aggregate printing technology, and their applications to tissue/disease models, artificial tissues, and drug-screening platforms. The paper also discusses the remaining hurdles and future directions of the printing processes.

The critical role of Hedgehog-responsive mesenchymal progenitors in meniscus development and injury repair.

Meniscal tears are associated with a high risk of osteoarthritis but currently have no disease-modifying therapies. Using a Gli1 reporter line, we found that Gli1+ cells contribute to the development of meniscus horns from 2 weeks of age. In adult mice, Gli1+ cells resided at the superficial layer of meniscus and expressed known mesenchymal progenitor markers. In culture, meniscal Gli1+ cells possessed high progenitor activities under the control of Hh signal. Meniscus injury at the anterior horn induced a quick expansion of Gli1-lineage cells. Normally, meniscal tissue healed slowly, leading to cartilage degeneration. Ablation of Gli1+ cells further hindered this repair process. Strikingly, intra-articular injection of Gli1+ meniscal cells or an Hh agonist right after injury accelerated the bridging of the interrupted ends and attenuated signs of osteoarthritis. Taken together, our work identified a novel progenitor population in meniscus and proposes a new treatment for repairing injured meniscus and preventing osteoarthritis.

Development of a decellularized meniscus matrix-based nanofibrous scaffold for meniscus tissue engineering.

The meniscus plays a critical role in knee mechanical function but is commonly injured given its central load bearing role. In the adult, meniscus repair is limited, given the low number of endogenous cells, the density of the matrix, and the limited vascularity. Menisci are fibrocartilaginous tissues composed of a micro-/nano- fibrous extracellular matrix (ECM) and a mixture of chondrocyte-like and fibroblast-like cells. Here, we developed a fibrous scaffold system that consists of bioactive components (decellularized meniscus ECM (dME) within a poly(e-caprolactone) material) fashioned into a biomimetic morphology (via electrospinning) to support and enhance meniscus cell function and matrix production. This work supports that the incorporation of dME into synthetic nanofibers increased hydrophilicity of the scaffold, leading to enhanced meniscus cell spreading, proliferation, and fibrochondrogenic gene expression. This work identifies a new biomimetic scaffold for therapeutic strategies to substitute or replace injured meniscus tissue. STATEMENT OF SIGNIFICANCE: In this study, we show that a scaffold electrospun from a combination of synthetic materials and bovine decellularized meniscus ECM provides appropriate signals and a suitable template for meniscus fibrochondrocyte spreading, proliferation, and secretion of collagen and proteoglycans. Material characterization and in vitro cell studies support that this new bioactive material is susceptible to enzymatic digestion and supports meniscus-like tissue formation.

Nuclear envelope wrinkling predicts mesenchymal progenitor cell mechano-response in 2D and 3D microenvironments.

Exogenous mechanical cues are transmitted from the extracellular matrix to the nuclear envelope (NE), where mechanical stress on the NE mediates shuttling of transcription factors and other signaling cascades that dictate downstream cellular behavior and fate decisions. To systematically study how nuclear morphology can change across various physiologic microenvironmental contexts, we cultured mesenchymal progenitor cells (MSCs) in engineered 2D and 3D hyaluronic acid hydrogel systems. Across multiple contexts we observed highly 'wrinkled' nuclear envelopes, and subsequently developed a quantitative single-cell imaging metric to better evaluate how wrinkles in the nuclear envelope relate to progenitor cell mechanotransduction. We determined that in soft 2D environments the NE is predominately wrinkled, and that increases in cellular mechanosensing (indicated by cellular spreading, adhesion complex growth, and nuclear localization of YAP/TAZ) occurred only in absence of nuclear envelope wrinkling. Conversely, in 3D hydrogel and tissue contexts, we found NE wrinkling occurred along with increased YAP/TAZ nuclear localization. We further determined that these NE wrinkles in 3D were largely generated by actin impingement, and compared to other nuclear morphometrics, the degree of nuclear wrinkling showed the greatest correlation with nuclear YAP/TAZ localization. These findings suggest that the degree of nuclear envelope wrinkling can predict mechanotransduction state in mesenchymal progenitor cells and highlights the differential mechanisms of NE stress generation operative in 2D and 3D microenvironmental contexts.

A study on the performance of the Donation Improvement Program in Korea.

Background: The Donation Improvement Program (DIP) is intended to increase organ donation at hospitals. The program includes education for healthcare professionals of participating hospitals about each step of donation as well as evaluation. The DIP consists of medical record review (MRR) and a Hospital Attitude Survey (HAS). The purpose of this study was to evaluate the DIP results for the last 7 years. Methods: For MRR, we analyzed 58,385 cases of mortality from 77 hospitals between 2012 and 2018. The HAS data for the degree of education experience, competence, and knowledge related to brain death (BD) and donation were analyzed from 23 DIP-participating hospitals in 2012 and 51 DIP-participating hospitals in 2015 and 2018 each. Results: The recognition rate of potential BD was 24.9%, 61.3%, and 73.2%, and donation rate was 7.5%, 11.7%, and 15.8% at 6 months before, 1?2 years after, and 4?5 years after the agreement, respectively. Hospital staff with the necessary competence or knowledge to explain BD constituted 44.0% in 2012, while this increased to 62.8% in 2018. Conclusions: The DIP could increase the recognition of BD and the organ donation rate, and positively affect the attitudes of healthcare professionals toward organ donation. Spreading the DIP to all hospitals is urgent to increase organ donation.

Decorin regulates cartilage pericellular matrix micromechanobiology.

In cartilage tissue engineering, one key challenge is for regenerative tissue to recapitulate the biomechanical functions of native cartilage while maintaining normal mechanosensitive activities of chondrocytes. Thus, it is imperative to discern the micromechanobiological functions of the pericellular matrix, the ~ 2-4 µm-thick domain that is in immediate contact with chondrocytes. In this study, we discovered that decorin, a small leucine-rich proteoglycan, is a key determinant of cartilage pericellular matrix micromechanics and chondrocyte mechanotransduction in vivo. The pericellular matrix of decorin-null murine cartilage developed reduced content of aggrecan, the major chondroitin sulfate proteoglycan of cartilage and a mild increase in collagen II fibril diameter vis-à-vis wild-type controls. As a result, decorin-null pericellular matrix showed a significant reduction in micromodulus, which became progressively more pronounced with maturation. In alignment with the defects of pericellular matrix, decorin-null chondrocytes exhibited decreased intracellular calcium activities, [Ca2+]i, in both physiologic and osmotically evoked fluidic environments in situ, illustrating impaired chondrocyte mechanotransduction. Next, we compared [Ca2+]i activities of wild-type and decorin-null chondrocytes following enzymatic removal of chondroitin sulfate glycosaminoglycans. The results showed that decorin mediates chondrocyte mechanotransduction primarily through regulating the integrity of aggrecan network, and thus, aggrecan-endowed negative charge microenvironment in the pericellular matrix. Collectively, our results provide robust genetic and biomechanical evidence that decorin is an essential constituent of the native cartilage matrix, and suggest that modulating decorin activities could improve cartilage regeneration.

Nutritional and Functional Properties of Fermented Mixed Grains by Solid-State Fermentation with Bacillus amyloliquefaciens 245.

Fermented foods have several advantages, including increased nutritional value, improved bioavailability, and functional health properties. We examined that these outcomes were also observed in fermented mixed grains (FMG) containing wheat germ, wheat bran, oats, brown rice, barley, quinoa, and lentils following solid-state fermentation (SSF) by Bacillus amyloliquefaciens 245. The metabolic profile during fermentation was screened using capillary electrophoresis time-of-flight mass spectrometry (CE-TOF-MS). The amino acids were quantitatively measured for the validation of the changes in metabolites. The activity of enzymes (e.g., amylase, protease, and fibrinolysis) and antioxidant capacity was also assessed to elucidate the functionality of FMG. The essential amino acid contents gradually increased as fermentation progressed. As the metabolites involved in the urea cycle and polyamine pathway were changed by fermentation, arginine was used as a substance to produce citrulline, ornithine, and agmatine. FMG showed dramatic increases in enzyme activity. FMG incubated for 36 h also displayed higher total phenolic contents and free radical scavenging ability than MG. The data suggest that FMG produced by Bacillus amyloliquefaciens 245 possess improved nutritional and functional quality, leading to their potential use as dietary supplements.

Nuclear softening expedites interstitial cell migration in fibrous networks and dense connective tissues.

Dense matrices impede interstitial cell migration and subsequent repair. We hypothesized that nuclear stiffness is a limiting factor in migration and posited that repair could be expedited by transiently decreasing nuclear stiffness. To test this, we interrogated the interstitial migratory capacity of adult meniscal cells through dense fibrous networks and adult tissue before and after nuclear softening via the application of a histone deacetylase inhibitor, Trichostatin A (TSA) or knockdown of the filamentous nuclear protein Lamin A/C. Our results show that transient softening of the nucleus improves migration through microporous membranes, electrospun fibrous matrices, and tissue sections and that nuclear properties and cell function recover after treatment. We also showed that biomaterial delivery of TSA promoted in vivo cellularization of scaffolds by endogenous cells. By addressing the inherent limitations to repair imposed by nuclear stiffness, this work defines a new strategy to promote the repair of damaged dense connective tissues.

Correction: Molecular subtypes of oropharyngeal cancer show distinct immune microenvironment related with immune checkpoint blockade response.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.