Whole organ tissue engineering encompasses a variety of approaches, including 3D printed tissues, cell-based self-assembly, and cellular incorporation into synthetic or xenogeneic extracellular matrix (ECM) scaffolds. This review article addresses the importance of whole organ tissue engineering for various solid organ applications, focusing on the use of extracellular (ECM) matrix scaffolds in such engineering endeavors. In this work, we focus on the emerging barriers to translation of ECM scaffold-based tissue-engineered organs and highlight potential solutions to overcome the primary challenges in the field. The 3 main factors that are essential for developing ECM scaffold-based whole organs are (1) recapitulation of a functional vascular tree, (2) delivery and orientation of cells into parenchymal void spaces left vacant in the scaffold during the antigen elimination and associated cellular removal processes, and (3) driving differentiation of delivered cells toward the appropriate site-specific lineage. The insights discussed in this review will allow the potential of allogeneic or xenogeneic ECM scaffolds to be fully maximized for future whole organ tissue-engineering efforts.  This review addresses the importance of whole organ tissue engineering for various solid-organ applications, focusing on the use of extracellular (ECM) matrix scaffolds in such engineering endeavors. In this work, we focus on the emerging barriers to translation of ECM scaffold-based tissue engineered organs and highlight potential solutions to overcome the primary challenges in the field. The insights discussed in this review will allow the potential of allogeneic or xenogeneic ECM scaffolds to be fully maximized for future whole organ tissue engineering projects.1-3 Donor organ shortage leads to ~15 waiting list patient deaths per day.2 While the short-term success rate of organ transplants has dramatically increased over time, complications related to immune rejection and the use of immunosuppressive drugs continue to limit long-term patient and/or graft survival.4-6 Whole organ tissue engineering aims to address the lack of organ availability by in vitro generation of viable human organs. Additionally, the use of autologous patient cells has the potential to overcome complications associated with rejection and use of immunosuppressive drugs in transplant recipients. Consequently, “off the shelf” tissue-engineered organs provide an enticing potential solution to overcome the limitations of allograft organ transplantation, by providing essentially unlimited organ supply while avoiding immune-mediated complications.Currently, approximately 110 000 patients are on the US organ transplant waiting list.7-12 Ongoing research continues to expand our knowledge of production methods, success criteria, and biological functions of unfixed ECM scaffolds toward increasing the scope and success of their clinical application. Acellular unfixed ECM scaffolds have attained widespread clinical use, with variable results depending on the application site, specific biomaterial, and processing method used.13-15 In cases where native ECM properties are retained, both allogeneic and xenogeneic ECM scaffolds have been shown to influence cell behavior, directing repopulating cell differentiation, proliferation, migration, phenotype, and function.17 This review focuses on the challenges associated with the use of xenogeneic ECM scaffolds in whole organ tissue engineering applications.Whole organ tissue engineering encompasses a variety of approaches, including 3D printed tissues, cell-based self-assembly, and cellular incorporation into synthetic, allogeneic, or xenogeneic extracellular matrix (ECM) scaffolds. Recapitulating the native structure, composition, and function of native tissue extracellular environment is challenging with synthetic, printing, or self-assembly-based tissue engineering approaches. By comparison, allogeneic or xenogeneic ECM scaffolds provide a potentially significant advantage in that they inherently possess the complexity of native tissue ECM structure/composition/function properties. The degree to which native tissue complexity is preserved varies depending on the specific antigen elimination/decellularization scaffold generation approach used.18 Recent work has made significant progress in defining specific antigenic barriers to xenotransplantation, exploring extraction conditions necessary to eliminate such antigens from candidate xenogeneic tissues, and establishing levels of antigen elimination required to achieve recipient graft-specific adaptive immune avoidance.19-21 Similarly, retention of native ECM structure, composition, and functional properties have all been shown to be instrumental in avoiding pro-inflammatory, while promoting pro-regenerative innate immune responses.12 The immunology of allogeneic or xenogeneic ECM scaffolds and strategies to overcome this challenge have been reviewed in detail elsewhere.22 As the challenge of producing immunologically acceptable acellular ECM scaffolds draws near accomplishment, research has increasingly shifted toward their utilization in tissue engineering applications. This review will focus on the emerging barriers to translation of ECM scaffold-based tissue-engineered organs and highlight potential solutions to overcome the primary challenges in the field. The primary barriers to the development of ECM scaffold-based whole organs are (1) Recapitulation of the entire vascular tree, which is essential to maintain the function of cells within the engineered organ and to allow communication with the central circulatory system. (2) Delivery and orientation of cells into the parenchymal void spaces left vacant in the scaffold during the antigen elimination and associated cellular removal processes. (3) Driving differentiation of delivered cells toward the appropriate site-specific lineage. Addressing these 3 primary obstacles will allow the potential of xenogeneic ECM scaffolds to be fully maximized for whole organ tissue engineering.The primary goal in generation of acellular ECM scaffolds is to eliminate antigenic tissue components while retaining the native tissue ECM structure, composition, mechanical properties, and biological function.23 In avascular constructs, as tissue thickness increases, nutrient and waste elimination requirements in the center of the graft outstrip diffusional supply, resulting in core necrosis.24 Although novel methods for overcoming diffusional distance limitations have been investigated in vitro , important barriers remain to their use in whole organ engineering.26 First, although microporous approaches can facilitate in vitro diffusion, fabrication of an appropriately organized hierarchical vascular tree to facilitate anastomosis to a recipient’s vasculature in vivo remains challenging. Furthermore, both hierarchy of the vascular tree and functional architecture of the native vascular wall are essential for regulation of organ perfusion, predominantly via arteriolar vasoactive responses.27 Finally, native vasculature also serves a critical role in regulating tissue hemostasis via endothelial cell (EC) and ECM  dependent mechanisms.28 Consequently, harnessing the native vascular structure, composition, and hierarchical organization of xenogeneic ECM scaffolds has potential to provide an ideal substrate for in vitro or in vivo whole organ perfusion, overcoming diffusional limitations to cell survival.The provision of vasculature is essential for whole organ tissue engineering to support cellular metabolic demands. This requirement stems from limitations of diffusion to sustain cell survival in engineered tissues.29-32 However, the effectiveness of varying approaches on maintaining native ECM structure-function properties depends on the particular chemical regime used. Such protocols have been applied via either diffusion  or perfusion  based protocols.34A wide range of decellularization or antigen removal approaches have been investigated and determined to be variably successful in generating acellular ECM scaffolds from candidate tissues and organs. Decellularization protocols utilizing detergents , sodium deoxycholate (SDC), and Triton-X 100), enzymatic digestion , and/or osmotic lysis  have been shown to effectively remove cells from a variety of tissues.36 The effect of various decellularization or antigen removal methods on resultant ECM structure is to some extent predictable, considering the mechanism of action of the compounds used during scaffold generation. The most commonly used decellularization compound  for example is a denaturing detergent. The denaturing effect of SDS combined with its high protein binding affinity result in the disruption of integral ECM proteins.38 SDS has been shown to intercalate in the triple helical structure of collagen, resulting in perturbation of collagen macromolecular structure, reducing scaffold collagen content, disrupting the elastin network, and altering resultant ECM scaffold functional properties.39-41 Use of the non-ionic detergent Triton X did not affect the structural integrity of the ECM components such as elastin and collagen II,29 but compromised collagen III content and degraded collagen IV integrity of the ECM. Decellularization of the scaffold using osmotic lysis was effective in removing endothelial cells, but ineffective in removing interstitial cellular elements.42 Furthermore, experience with SynerGraft heart valves suggests that hypotonic lysis alone results in inadequate elimination of antigenic components to overcome in vivo graft-specific adaptive immune responses, with severe adaptive immune destruction of the valve biomaterial reported in human patients.43 Both non-specific  and targeted  enzyme-based protocols have been used in decellularization protocols to reduce biomaterial antigen content.45 As might be expected, off-target disruption of structural ECM proteins with non-specific enzymatic approaches has generally been reported to have more detrimental effects on the resultant ECM than that seen with targeted enzymatic approaches.45 For instance, non-specific enzymatic digestion led to distortion and fragmentation of elastic fibers present on the ECM, resulting in altered mechanical behavior and reduced extensibility by increasing the scaffold stiffness.46 Conversely, targeted enzymatic antigen elimination approaches  are effective in reducing individual antigen content and avoiding off-target damage to the remaining ECM.44 However, targeted enzymatic approaches are frequently combined with other approaches, since they fail to eliminate cellular or antigenic components that are not associated with the primary enzymatic target. For instance, Wu et al optimized SDS decellularization of an acellular porcine annulus fibrosus scaffold by including α-Gal epitope reduction  using alpha-galactosidase administration.48 Consequently, consideration of the mechanism of action of chemicals used in decellularization/antigen removal approaches and their effect on the resultant biomaterial are critical considerations in ECM scaffold generation.Treatment of whole organs with perfusion-based decellularization has had varying effects on the resultant ECM and associated vascular structures.36 Some groups have demonstrated intact vascular structures and successful perfusion recellularization of vessels as small as 10 µm.49 However, cellular repopulation or transit across capillary beds to the venous vasculature was reported to be infrequent, likely due to disruption of at least some portions of the capillary bed during the decellularization process.35 Even in instances where the capillary-like structures were identified following perfusion seeding of rabbit whole heart scaffolds with human induced pluripotent stem-cell derived endothelial cells, the number of repopulated capillary-like structures was extremely small compared to the expected number for myocardial tissue .49 Similarly, successful dye transit across arterial to venous vascular beds has also been reported.51 While these results suggest the potential for intact capillary beds, it is also possible that leakage into the scaffold parenchyma may occur from smaller vessels, and then re-join the venous vascular tree at larger venules/veins, giving the impression of an intact vascular network , distal vessels in the vascular tree of solid organs are largely disrupted  by reported decellularization approaches. network .35 Conse53 Recent studies utilizing ECM scaffolds have demonstrated the potential for BM components  to modulate repopulating EC phenotype, function, migration, and monolayer formation.55 Critically, BM components have been reported to prevent Z-direction  migration of seeded cells. In the case of ECs, the capacity of BMs to prevent Z-direction migration has potential to guide to facilitate cellular repopulation of the entire vascular network .54 However, the delicate structure of the BM has proven extremely challenging to maintain in decellularized ECM scaffolds. For instance, Faulk et al examined the effect of 4 different detergent-based decellularization methods  on BM integrity.34 The authors demonstrated disruption of BM components  using all decellularization methods, although the extent of disruption varied with the detergent used. Triton X-100 and sodium deoxycholate treated scaffolds retained some staining for Col IV for instance, which may explain the compatibility of these scaffolds with human EC repopulation and monolayer formation, although laminin and Col VII disruption were extensive.34 Similarly, Schneider et al demonstrated disruption of placental artery BM integrity following either SDS or Triton X-100 decellularization.56 Alternatively, Shupe et al reported preservation of laminin in large diameter vessels of whole liver scaffolds formed using Triton X-100 followed by 0.1% SDS decellularization.57 However, capillary laminin staining and Col IV presence were incompletely preserved. Finally, Calle et al used a proteomic method to show that lung scaffolds decellularized with a “gentle” Triton X-100, sodium deoxycholate method preserved 75% of the analyzed BM proteins at near-native levels.58 As expected, preservation of BM components enhanced endothelial and mixed lung cell repopulation of the resultant scaffold following perfusion-based seeding. However, this “gentle” approach resulted in increased retention of cellular proteins compared to traditional CHAPS decellularization methods.58 Conversely, use of the non-denaturing non-ionic detergents  or ASB-16) in antigen removal methods has been reported to eliminate antigenic components while retaining native tissue BM components .59 Importantly, such antigen removed scaffolds have been shown to modulate appropriate quiescent monolayer formation of repopulating ECs, mirroring the behavior identified in single component hydrogel studies.59 Consequently, ongoing refinements of whole organ ECM scaffold production methods have potential to leverage BM integrity combined with perfusion-based EC seeding toward recapitulation of the native vascular tree.The ubiquitous production of basement membrane (BM) components by vascular ECs provides a marker of vascular network integrity and important functional cues to guide repopulating cell behavior. The importance of individual BM components has been well established in hydrogel models in vitro and reviewed elsewhere.60 Indeed, publications examining coculture of ECs and pericyte precursors in the vascular compartment of whole organ ECM scaffolds have shown improvement in the extent of endothelialization compared to previously reported monoculture systems  as a source for EC’s, and is often co-seeded with human mesenchymal stem cells (hMSC) to serve as a perivascular cell population.61 Perivascular support from hMSCs was shown by Ren et al to improve EC monolayer coverage compared to EC seeding alone .61 Furthermore, other experiments have shown that co-seeding other cell types alongside endothelial cells can improve cellular function.62 Finally, continuous perfusion with seeded endothelial or endothelial progenitor cells has also been able to regenerate a relatively evenly distributed endothelium throughout whole organs.62-64 Consequently, although this work suggests that coculture and continuous perfusion may improve vascular endothelialization, future work focused on the proportion, sequence of delivery, duration of seeding, and culture conditions required to optimize the outcome for each cell type will be required to further improve vascular function in ECM scaffold-based tissue engineered constructs.The native vascular compartment is comprised of EC, vascular smooth muscle cells (vSMC’s), and perivascular MSC’s (pericytes). While all cell types are required for healthy vascular function, the importance of a quiescent EC monolayer in modulating vascular tone, coagulation state, platelet adhesion, barrier function, and leukocyte adhesion has led the majority of publications to focus on EC repopulation of whole organ grafts. However, the importance of other cell types, particularly pericytes, in maintaining healthy EC function has recently been appreciated. systems .61,62 A 75-78 Some of these studies also investigated varying perfusion flow rates at different post-seeding times, suggesting that an increase of physiologic mechanical signals toward normal physiologic forces may drive complete monolayer coverage and endothelial cell maturation.79-82 This mimicking of physiologic conditions aims to gradually provide native mechanical stimuli and promote cellular function which approximates that seen in vivo. Overall, the current evidence supports the conclusion that a gradual increase in flow toward physiologic values results in enhanced cell adhesion, invasion, and proliferation. Future work is needed to determine the optimal timing, flow rate, modality , and viscosity of perfusion to maximize in vitro and resultant in vivo tissue-engineered vascular health.In addition to determining the optimal delivery of cell types to achieve construct revascularization, consideration must also be given to maintaining the health and function of these cells. Since almost all whole organ recellularization investigations are undertaken in vitro, the use of bioreactors to generate a specific set of conditions conducive to cell health is ubiquitously used. The effect of shear stress on endothelial development has been widely investigated, with custom-made bioreactors used to examine the effect of laminar versus pulsatile flow, both with mixed results.83-86 Progress in cellular repopulation of ECM scaffolds must consider the route of cell seeding, guidance of cell migration within the scaffold, and the cell type/types with which to seed the scaffold to achieve maximal repopulation.Recellularization of ECM scaffolds with parenchymal cells is essential to establish physiologic function of engineered tissues and organs. Physiologic activity of all tissues and organs is critically dependent on cellular composition, orientation, interactions , and function. To date, recapitulation of such sophisticated cellular architecture and activity has been hindered by our fundamental inability to achieve cellular repopulation of more than a fraction of the parenchymal void spaces created via primary cell removal during ECM scaffold production.87-89 As previously discussed, acellular ECM scaffolds have already achieved clinical use in relatively planar applications, where short diffusion distances allow rapid in vivo cellular repopulation supported by later angiogenesis. Although such in vivo bioreactor approaches may be capable of restoring the partial function of larger organs ,90-94 implantations of acellular ECM scaffolds is unable to provide immediate functional benefit for critical organ defects  or whole organ replacement  applications. Although in vivo cellular repopulation of parenchymal void spaces demonstrates the feasibility of generating functional tissue from ECM scaffolds, engineering whole organs with immediate post-implantation function requires in vitro recellularization. Further investigation of the mechanisms responsible for in vivo cellular parenchymal void space repopulation in ECM scaffolds is necessary to inform ongoing attempts to recapitulate such processes in vitro  surface seeding, (2) intra-scaffold injection seeding, and (3) perfusion seeding  or type I collagen  containing ECM niches. As previously discussed, BM components have been shown to represent a potent barrier to Z-direction migration of both endothelial and mesothelial cells.101 Ott et al decellularized rat hearts by coronary perfusion with detergents, preserved the underlying extracellular matrix, and produced an acellular, perfusable vascular architecture, competent acellular valves, and intact chamber geometry.33 Perfusion decellularized scaffolds were reseeded with intramural injection of neonatal cardiac cells and perfusion of rat aortic endothelial cells into the vascular conduits. The resulting heart constructs showed contractile responses to electrical pacing stimuli and were responsive to phenylephrine drug administration on day 8 of culture. Recellularization was greatest (33.8 ± 3.4% recellularization) in the area of injection . By day 8, there were areas of confluent cellularity approximately 1 mm thick and viability was >95% throughout the entire thickness (0.5-1.1 mm). However, recellularization rapidly diminished at remote sites distant from the injection . Although this landmark publication demonstrated the potential benefits of perfusion-based decellularization, the extent to which injection-seeded primary cells migrated into and oriented with the parenchymal void spaces remains unclear. Additionally, since multiple different cell types were delivered in each injection  the influence of each cell type in achieving the observed confluent cellularity is difficult to assess. Shirakigawa et al compared the efficiency of void space repopulation following recellularization of decellularized rat livers with primary rat hepatocytes delivered via parenchymal injection, hepatic view perfusion, or portal vein perfusion. The authors found that hepatocyte perfusion resulted in clogging of the vasculature with primary hepatocytes, with minimal migration of the cells into the parenchymal spaces. Parenchymal injection resulted in significantly greater void space repopulation and enhanced hepatocyte function . However, as seen in other injection seeding publications, void space repopulation was largely confined to the site of injection. The above results demonstrate that it is difficult to know if the cells simply proliferate in situ at the injected site, without truly integrating with the scaffold, or migrate into the ECM void spaces and fully integrate with the scaffold following injection seeding. Consequently, future studies are required to clearly define the migratory potential of various injected cell types, characterize the extent of cellular integration within the ECM , and determine the capacity of the ECM to modulate the orientation of seeded cells within the parenchymal void spaces. Even if injection-seeded cells are able to migrate and integrate with the ECM void spaces of the scaffold, delivery of a large enough number of cells via this approach to repopulating all void spaces remains challenging.Injection of cells directly into ECM scaffolds attempts to overcome the limitations of surface seeding by delivering cells directly to the ECM niche of interest.102-104 However, as discussed in the vascularization section, perfusion-based seeding approaches are critically dependent on preservation of native tissue ECM integrity and associated niche properties. Disruption of vascular beds during scaffold preparation may allow perfused cells to leak into parenchymal void spaces, creating the false appearance of void space repopulation .pulation . Consequ69 Petersen et al used intratracheal perfusion-based seeding of a mixed population of neonatal rat lung epithelial cells, resulting in cellular repopulation of alveolar structures, as well as small and medium-sized airways.95 Microvascular lung endothelial cells, were also injected into the pulmonary artery of acellular scaffolds which adhered throughout the scaffold vasculature. Similarly, Ott et al studied the repopulation of the lung with HUVECs (via vascular perfusion) and carcinomatous human alveolar basal epithelial cells (cell line A549) via transtracheal perfusion.91 This approach led to dispersion of endothelial cells throughout the entire pulmonary vascular tree. Simultaneously, epithelial cells showed engraftment and formation of an epithelial monolayer after 5 days in culture throughout the entire lung construct, with preservation of parenchymal architecture. Consequently, perfusion-based seeding approaches show clear promise for increasing the number and distribution of delivered cells in ECM scaffold void spaces, although current progress in solid organs lags behind that of organs containing multiple potential perfusion routes .In the case of lung perfusion recellularization, vascular perfusion has been used to deliver endothelial cells to the vascular bed, while airway perfusion has been used to deliver epithelial cells to the respiratory tree.106 Furthermore, compaction of the ECM may occur due to loss of primary cell pre-stress on the ECM following decellularization further compromising seeded cell migration. Furthermore, specialized ECM proteins  are known to serve as barriers to cell migration  and must therefore be considered in attempts to drive void space migration and repopulation.99 Consequently, ECM porosity, composition , cell migration capacity, and cellular function  should all be taken into account when designing solid-organ perfusion recellularization approaches.For solid organs, assuming perfect maintenance of ECM structure throughout the decellularization process, no perfusion route exists which directly communicates with the parenchymal void spaces. Consequently, repopulating cells are required to migrate from the perfusable vascular tree , through the interstitial ECM (predominantly type I collagen in the majority of organs), and ultimately into the parenchymal void spaces . To achi107Given the challenges inherent to achieving Z-direction migration of perfusion-seeded cells across the vascular BM, through the interstitial ECM and into void spaces, it is unlikely that full repopulation can be achieved without providing some external factors to influence the proportion, direction, and speed of cell migration through ECM scaffolds. Consequently, a number of groups have recently focused on determining the mechanisms by which in vitro XY  and Z direction cell migration throughout ECM scaffolds can be controlled. Signals for cell migration maybe soluble , matrix bound , or mechanical .108 basic fibroblast growth factors ,109 vascular epithelial growth factor,110 and transforming growth factor-beta (TGF-β).111 GFs control a wide range of biological functions, including cell proliferation, survival, migration, and differentiation. For example, Leuning et al bioengineered re-endothelialized whole kidney matrix scaffolds from both rodents and humans using human inducible pluripotent stem cell-derived endothelial cells along with vascular endothelial growth factor and angiopoietin 1 while achieving efficient cell delivery, adherence, endothelial cell proliferation, and survival.67 The processes of cell migration, differentiation, and proliferation are typically dependent on both the presence of specific growth factors and their spatial- and time-dependent distributions. Consequently, the most critical issue related to growth factors is delivery of physiologically relevant doses and control of the duration of action of such molecules at the site of action due to their burst release kinetics and localized concentration following injection-based delivery strategies.112 To overcome the deficiencies of direct injection, several ways of GFs delivery have been developed such as cell transfection with DNA plasmids encoded with the desired GF, introducing an inducible GF gene into the cellular genome (gene editing) and implantation of GF proteins directly into the cell.114In the case of soluble signals, the most commonly used growth factors (GFs) have included bone morphogenic proteins (BMP),115-118 Although these studies have demonstrated successful control of cell migration, they have predominantly used hydrogel-based scaffolds, while controlled recellularization of decellularized ECM scaffolds through chemotactic gradients has not been widely reported. Furthermore, as previously discussed, poor void space filling deep within tissues presents a challenge; directing migration into tissues (Z-direction) is much more difficult than migration across scaffold surfaces (X- or Y-direction), at least in part because of the fact that BMs present a barrier to Z-direction migration.59 Some groups have achieved early success by using external factors to influence Z-direction migration of cells into the matrix. Pre-treating scaffolds with exogenous factors  have been used to accelerate initial cell migration into decellularized ECM scaffolds.119 Assmann et al demonstrated that fibronectin coating was successful in accelerating autologous in vivo recellularization of aortic conduits following implantation in Wistar rats for up to 8 weeks. Fibronectin coating resulted in significantly accelerated luminal endothelialization (P = .006 after 8 weeks), and enhanced myofibroblast hyperplasia, significantly increasing the intima-to-media thickness ratio (P = .0002 after 8 weeks). Alpha-smooth muscle actin and desmin positive cell invasion into the vascular media of fibronectin-coated conduits originating from the adventitial surface was significantly increased as compared to uncoated control conduits (P < .0001). This work shows that biofunctional protein coating accelerated in vivo endothelialization and induced a significantly increased medial recellularization, however, the effects of such pre-treatment did not last and still resulted in incomplete void space repopulation deeper in the tissue.119 Additionally, despite the promise of such ECM coating approaches, dose-dependent control of such stimuli must be achieved to avoid potential negative consequences . Further research into the manipulation of chemotactic gradients in vitro is needed before an optimal combination of seeding method and culture conditions is established. Achieving control of cellular migration in ECM scaffolds could enable the complete repopulation of void spaces, a goal that has evaded tissue engineers for some time. Others have investigated nutrient sources in the perfusing media as a driver of cellular migration.120 Tosun et al aimed to enhance cellular migration by applying directed mechanical and nutritional gradients to promote transmural migration and more uniformly populate vascular constructs. Using perfusion bioreactors, lumen pressure (either 120/80 mmHg [high pressure] or 50/30 mmHg [low pressure]) was used to modulate transmural hydraulic conductivity, either with, or without, media in the abluminal flow circuit. Cells seeded on the abluminal surface migrated toward the lumen when media was perfused in the luminal flow path only, regardless of perfusion pressure. However, the extent of migration was maximized when low-pressure perfusion (SP-Lp: 50/30 mmHg) was used compared to the high-pressure perfusion group. Both of the above studies use vascular scaffolds, which easily enable separate control of the luminal and abluminal conditions. This method allows easy and effective establishment of chemotactic and/or transmural pressure gradients, but translation of these principles to whole organs may present challenges since isolation of the perfusable lumen from the parenchymal void spaces is more challenging in such geometries.Chemotactic gradients can be applied to encourage cell migration across scaffolds, employing a variety of matrix materials as well as cell types.121 Recent studies have revealed the importance of biomechanical conditions in addition to biochemical cues for cell signaling and migration.122 It has been suggested by Zhao et al, that biochemical cues are better at guiding cell migration with improved directionality and persistence, while mechanical cues are better at coordinating collective cell migration.123 Extent to which such reductionist approaches could be applied to manipulate the properties of naturally derived ECM scaffolds remains underexplored and may represent an area for future advances.Cell migration is also influenced by matrix factors such as stiffness and porosity. This is particularly important with scaffolds derived from natural tissues where pore size is subcellular limiting cell seeding and adhesion to the scaffold periphery. The density and spatial alignment of the scaffold determine the mechanical tissue properties , which guide or oppose cell migration and positioning. Wolf et al expressed that the structural characteristics of connective tissue-derived collagen display a dual function for cell movement as (1) guidance structure providing preformed tracks, and (2) barrier functions by forming fiber networks with random gaps of subcellular size.124 This necessity drives the need to understand and control in situ differentiation of such stem/progenitor cells following parenchymal void space repopulation. While the previously mentioned challenges of void space repopulation have hindered progress in the ECM scaffold-driven cell differentiation field, some limited successes have been reported. Early attempts to examine the potential for ECM scaffolds to modulate cell differentiation used micronized  ECM scaffolds  which may be capable of driving cell differentiation. Insights from reductionist approaches indicate that many of the signals which are capable of stimulating migration  are also capable of influencing cell differentiation or maintaining primary cell differentiating. For example, Kudva et al showed that primary human articular chondrocytes (hAC) de-differentiate when grown on 2D surfaces, but 3D polyethylene glycol (PEG) hydrogels incorporated with RGD maintained hAC viability and chondrogenic gene expression over time.125 Similarly, Flaim et al explored the feasibility of using ECM protein microarrays to study both primary rat hepatocyte dedifferentiation and embryonic stem (ES) differentiation toward early hepatic lineage. Combinations of ECM proteins were identified that were capable of driving ES differentiation and preventing de-differentiation of primary cells.126 Consequently, there is evidence that ECM components are capable of driving stem cells and maintaining terminal cell differentiation. The development of perfusion decellularization and recellularization approaches facilitated the study of site-specific  differentiation potential of intact ECM to be studied.Although it is possible for tissue engineering efforts to employ primary cells in some settings  lack of migratory capacity, limited availability, and/or poor proliferation capacity of many primary cell types  drive the need to employ progenitor and/or stem cells for parenchymal void space seeding in many solid-organ engineering applications.caffolds . For ins127 MSCs have received particular attention due to their tri-lineage differentiation potential and immunomodulatory properties, which may have benefits in modulating residual ECM scaffold-specific immune responses.128 However, the multipotent potential of MSCs proved to be a major limitation to their use in whole organ engineering efforts, since they were incapable of differentiation to many of the required primary parenchymal cell types. Unlike MSCs, human embryonic stem cells (hESCs) derived from the inner core of blastocytes have pluripotent capacity. This benefit has led to use of hESCs in a number of recent publications.129 For example, Rajabi et a generated decellularized rat hearts, with or without immobilized basic fibroblast growth factor (bFGF), followed by perfusion reseeding with human embryonic stem cells (hESCs)-derived cardiovascular progenitor cells (CPCs). bFGF immobilization improved retention of CPCs, with cells demonstrating ECM site-specific differentiation toward cardiomyocyte, smooth muscle cell, and endothelial cell lineages.72 However, although parenchymal void space repopulation reached 50.2% in the LV free wall, the remainder of the myocardial tissue was less well populated (~24% void space filling). Despite the current technical limitations of parenchymal void space repopulation, such studies demonstrate the potential of ECM scaffolds to inform site-specific cell differentiation.Due to the limited supply of autologous primary cells and their phenotypic instability following in vitro proliferation, stem cell plasticity in tissue culture has been widely explored. For this purpose, multi- or pluripotent stem cells were isolated from various regions of the body, ranging from tissue-specific progenitor cells, to circulating progenitors and mesenchymal stem cells (MSC).130 Alexanian et al used perfusion-based seeding of decellularized mouse heart ECM scaffolds with induced cardiac progenitor cells (iCPC) reprogrammed from either lung or cardiac fibroblasts.74 Following seeding the ECM scaffold drove iCPCs differentiation toward cardiomyocytes, endothelial, and smooth muscle lineages in a site-specific manner .74 The differentiated cardiomyocytes showed an increase in cell size and sarcomere organization compared to in vitro differentiated iCPCs, suggesting that in addition to the growth factors and small molecules that were perfused to promote differentiation of iCPCs, the appropriate cardiac ECM niche further facilitated differentiation. Despite these promising results, the proportion of cardiomyocyte to endothelial cell differentiation was far from that expected for native cardiac muscle. Similarly, Hochman-Mendez et al used sequential perfusion seeding with human iPSC-derived endothelial cells and cardiomyocytes to repopulate decellularized rabbit whole heart ECM scaffolds.49 Seeded cells regenerated left ventricular (LV) wall to full thickness from base to apex and endocardium to epicardium. However, cellular repopulation of the interventricular septum and right ventricular free wall was less successful. Although cardiac and endothelial cell site-specific differentiation was observed, as with other similar studies the ratio of endothelial cells to cardiomyocytes was far lower than expected for native cardiac muscle. Consequently, although this study demonstrated the potential for ECM to inform iCPC cell differentiation in a site-specific manner, future work is needed to further understand and optimize the ECM factors responsible for driving such differentiation.Development of induced pluripotent stem cells (iPSC) has further fueled interest in in vitro ECM scaffold cellular repopulation and differentiation studies.131 The effects of this scaffold on cell shape, cytoskeleton organization, cell viability, and osteogenic differentiation were examined. The results indicated that periodontal ligament stem cells cultured on HAp-PADM exhibited different cell shapes when compared with those on pure PADM. Moreover, HAp-PADM promoted cell viability and increased alkaline phosphatase activity significantly. It was concluded that the surface HAp nanonetwork structure of PADM could promote the viability and differentiation of PDLSCs and is a promising scaffold for periodontal tissue engineering.131 However as for non-functionalized scaffolds this field is hindered by the incomplete void space repopulation issue which results in cells that may or may not be interacting with native ECM across their entire surface.Functionalization of ECM scaffolds to further enhance cell function/differentiation has also been studied. For example, Shaohua Ge et al fabricated a nanostructured hydroxyapatite-coated porcine acellular dermal matrix (HAp-PADM) by a biomimetic mineralization method and seeded human periodontal ligament stem cells (PDLSCs).Consequently, although current studies show that the ECM has potential to drive cell differentiation beyond embryonic stages, several barriers exist which currently prevent the full potential of this field from being realized. Solving the parenchymal void space repopulation issue is the first critical barrier to progress since without ensuring correct orientation of repopulating cells within the 3-dimensional matrix the signals they receive will at best be “diluted” . Even if the void space issue can be solved, future work focusing on the effect of scaffold preparation techniques on ECM-bound growth factors, matricryptic signal, matrikines, and mechanical properties and their effects on cell migration, proliferation, function, and differentiation will be required before the full potential of ECM scaffolds can be harnessed.


Marine heatwaves cause widespread environmental, biological, and socio-economic impacts, placing them at the forefront of 21st-century management challenges. However, heatwaves vary in intensity and evolution, and a paucity of information on how this variability impacts marine species limits our ability to proactively manage for these extreme events. Here, we model the effects of four recent heatwaves  in the Northeastern Pacific on the distributions of 14 top predator species of ecological, cultural, and commercial importance. Predicted responses were highly variable across species and heatwaves, ranging from near total loss of habitat to a two-fold increase. Heatwaves rapidly altered political bio-geographies, with up to 10% of predicted habitat across all species shifting jurisdictions during individual heatwaves. The variability in predicted responses across species and heatwaves portends the need for novel management solutions that can rapidly respond to extreme climate events. As proof-of-concept, we developed an operational dynamic ocean management tool that predicts predator distributions and responses to extreme conditions in near real-time. This study examines the effect of four marine heatwaves in the Northeast Pacific on the distributions of 14 top predators, revealing a wide-array of predator responses both among and within heatwaves. Predator responses were highly predictable, demonstrating capacity for early warning systems of heatwave impacts, similar to weather forecasts. The impacts of long-term climate trends have received considerable attention through the examination of the warming signal in both historical observations and future climate projections9. However, mounting evidence indicates that episodic events like fires, floods, and heatwaves can have catastrophic ecosystem and socio-economic impacts12. In particular, heatwaves on land13 and at sea1 adversely impact an additional 157 million people today compared to the turn of the century14. The largest and warmest marine heatwaves (MHWs) on record have occurred in the last decade and include the 2012 Northwest Atlantic15, the 2015–16 Tasman Sea16, and 2013–16 Northeast Pacific17 events, exerting considerable physical, ecological, socio-economic, and human health impacts1.Long-term climate trends  and short-term extreme events  have global impacts on ecosystem structure and functioning, and human well-being2. This phenomenon is well-described within the context of long-term warming8 and El Niño events19, yet there is a paucity of information on species redistribution in response to unprecedented warming during recent MHWs20. Temperatures observed during MHWs can be similar to projected mean future conditions at the end of the 21st century21, providing valuable insight into species redistribution in the coming decades. Previous investigations on MHW-driven species range shifts have largely relied on observational data from opportunistic sightings, surveys, or tagging programs, and have demonstrated poleward or vertical distributional shifts towards cooler temperatures26. However, these data are often patchy, offering only snapshots of impacts from a single MHW on a single species27 or on several species28. Comparisons of multi-species responses across multiple MHWs are rare despite ample evidence that there is high variability in the evolution, drivers, and physical characteristics of MHW events30. Statistical models provide a means of interpolating across space, time, and taxa, providing information on MHW-driven redistribution by offering inferences on unobserved locations, MHWs, and individuals. Furthermore, statistical models can relate species distributions to multiple environmental drivers, thereby accounting for the complex physical and biogeochemical changes beyond the increased temperature that occurs during MHWs29.Faced with a changing climate, mobile species’ first responses are often to shift their geographic ranges to remain within suitable environmental conditionsHere, we quantify the impacts of four major North Pacific MHWs  lost and gained predator habitat and where adaptable governance strategies are likely to be important. By implementing a multi-species, multi-MHW analytical framework, our work provides the basis for informing future management of the impacts of these extreme climatic events.The Northeast Pacific Ocean provides an ideal testbed for examining the effects of MHWs. This region has experienced some of the longest, largest, and most intense MHWs on historical record32 Fig. , with thn33 Fig. . These p37 to predict the redistribution of species’ preferred habitats during each MHW event. Our models accurately predicted distribution shifts during MHW years captured by an extensive (>one million records) independent top predator dataset collated from public, private, and government sources  may be greater, as these species are less capable of accessing distant patches of suitable habitat. The models captured species fundamental niches, the full set of environmental conditions where a species foraged or transited within the North Pacific. However, they did not account for their environmental preferences during reproductive behaviors  or other ecological processes that may affect distribution 45.Correlative species distribution models such as those used in the present study are valuable tools to understand species ecology and are often used to support environmental conservation and management across multiple time horizons5. Here, we demonstrate that MHWs may drive jurisdictional shifts over more immediate time-frames, leading to new national risks, rewards, and responsibilities. During each event, our models predicted an influx of species habitat into the US EEZ from the Mexican EEZ and the high seas  wars47, offering precautionary tales for the value of coordinated and proactive management across nations.Long-term warming has redistributed species across EEZ boundaries, and is projected to continue to redistribute species in the futureeas Fig. . Predato25. These episodic fishing opportunities will require rapid managerial oversight to ensure stock sustainability, e.g., a climate-driven tilefish redistribution along the US east coast led to the stock being exploited without regulation for nearly a decade5. Predicted core habitats of protected species also shifted into US waters: elephant seal and leatherback turtle habitats were redistributed to the US EEZ from the high seas. Both species are bycaught in US fisheries , and may require increased monitoring to ensure fisheries interactions do not threaten their populations. Protected blue whale habitat shifted from Mexican to US waters, potentially increasing management concern over mortality from ship-strikes and entanglement in fishing gear which has been observed in US waters during MHW conditions49. Up to 39% of threatened white shark habitat shifted into the US waters from the high seas—the largest redistribution of any species  during anomalous conditions. Importantly, these new observations could be assimilated with distribution model outputs to improve real-time model predictions, a common process in oceanography, which is not yet a standard practice in ecology53.Dynamic ocean management55, and skillful forecasts of MHWs have recently become operational at lead times of up to a year, depending on the region56. The next frontier is to skillfully forecast ecological variables, including species distributions, in response to these extreme episodic events. These early warning systems would allow for proactive—as opposed to reactive—responses to new human-wildlife conflicts, changing marine resource availability, and emergent refugia caused by MHWs57, allowing us to plan ahead for our fundamentally dynamic world.Forecasts of future impacts would offer longer lead times for decision-making compared to real-time predictions. Extreme episodic events like hurricanes, fires, and floods are successfully forecast on land33 and private datasets. For all species except albatrosses, detailed methods on the number of individuals tagged, handling of tagging bias, and state space modeling are included in Block et al.33, Winship et al.36, and Jordan et al.37. Further details on albatross telemetry data are available in Supplementary methods section Telemetry data used to build the species distribution models (SDMs) were acquired for 14 top predators tagged from 2000–2010, including data from the Tagging of Pacific Predators ProjectPrionace glauca] and mako [Isurus oxyrinchus] sharks; albacore [Thunnus alalunga], yellowfin [T. albacares], and bluefin [T. thynnus] tunas; blue whales [Balaenoptera musculus], sooty shearwaters [Ardenna grisea], and California sea lions [Zalophus californianus]), despite some of these species undertaking long migrations across the North Pacific. Northern species had observations to the northwest of Coastal species ; and Southern species had observations to the southwest (white sharks [Carcharodon carcharias] and leatherback turtles [Dermochelys coriacea]). These regional groupings were useful to describe broad patterns, as MHW impacts were most similar across species within the same geographical group. The 75th percentile kernels for each geographical group were calculated by randomly subsampling each species’ locational data to the same number of records, and then calculating kernel densities and the 75th percentile kernel across all species within each grouping  models used the multi-year animal telemetry data to describe species-environment relationships from a suite of environmental variables , particularly during MHW years  as these conservative thresholds sometimes resulted in a complete loss of core habitat during extreme events .The continuous habitat suitability predictions were reclassified into daily binary core habitat using species-specific thresholds . To quantify species’ range extents, the daily north-south interquartile range was multiplied by the daily east-west interquartile range . The interquartile range was selected over the absolute range to remove the effect of outliers , and to ensure that expansion of range was not artificially truncated due to proximity to the convex hull boundary.Three metrics were calculated from the daily binary core habitat rasters: the center of gravity of core habitat (mean latitude/longitude coordinate pairs), the interquartile range of core habitat (latitude/longitude coordinate pairs for the north-south and east-west interquartile ranges), and the total amount of core habitat , and deviations from the 2000–2020 August–October mean was calculated for each habitat metric and each species, producing estimates of habitat displacement, changes to the range extent, and changes to the habitat area. Habitat displacement was assessed as a vector  joining the long-term mean position of the core habitat center of gravity to its MHW-specific location. Range extent change reflected the MHW-specific compression or expansion of range extent compared to the mean range, measured by area, while habitat area change assessed the MHW-specific loss or gain of total core habitat, measured by area. In addition to species-specific habitat metrics . In order to make MHW impacts comparable across species, range compression/expansion and habitat area loss/gain were expressed as a percent change from mean conditions.59  boundaries were used to define national jurisdictions (marineregions.org)Supplementary InformationPeer Review FileSource Data


This paper examines the linkage between Chinese stock market volatility and investor attention fluctuation. In Heterogeneous autoregressive (HAR) model, first, we analyzed the linkage between both decomposed and undecomposed stock market realized volatility and investor attention fluctuations across full-sample and two-year moving window sub-samples. Second, we compare the predictive power of four models in short-, medium-, and long-term volatility forecasting. Empirical results show large positive attention fluctuation amplified Chinese stock market volatility after the outbreak of COVID-19, and negative small attention fluctuation significantly stabilized stock market volatility before COVID-19, and the impact dwindled in after COVID-19. The model incorporating decomposed realized volatility and decomposed attention fluctuation performs better in volatility Forecasting. This research underscores a shift in the dynamics between stock market volatility and investor attention fluctuations, and investor attention fluctuation improves the volatility forecasting accuracy of the Chinese stock market. Each oscillation within the stock market can be attributed to the psychology of investors. The stock market, serving as a platform for capital distribution, reflects a nation’s political, economic, and cultural landscape. Although Chinese stock market ranks as the second largest market globally, it is predominantly comprised of individual investors. The attention fluctuations within this group of investors can significantly influence market dynamics. Therefore, to invigorate the capital market and foster sound development of the Chinese stock market, it is crucial to augment the exploration of the relationship between investor attention fluctuation and market volatility in Chinese stock market.Simon  introducAndrei and Hasler  pioneereCOVID-19 pandemic has elicited a range of economic responses. Bao and Huang  highlighThe complexity of stock market dynamics, especially in the face of external shocks like the COVID-19 pandemic, underscores the need to delve deeper into the factors influencing stock market volatility. We primarily investigate the impact of fluctuations in investor attention on the realized volatility (RV) of the Chinese stock market. To address this, we develop an attention index derived from Baidu search volume data and conduct a comprehensive analysis of the range of investor attention fluctuations. We categorize investor attention fluctuations into four levels—small, medium, large, and major—based on varying percentage changes in the attention index.Regarding contributions of this paper, by estimating the relationship between investor attention fluctuation and stock market realized volatility with HAR model, we can find that positive attention fluctuations exceeding 7% become pivotal in affecting Chinese stock market volatility post the outbreak of COVID-19. Negative attention fluctuations below 3% significantly impact stock market volatility before COVID-19, but this effect dwindles in post the outbreak of COVID-19 pandemic. Moreover, the incorporation of the decomposed investor attention volatility into the model enhances the accuracy of short and medium -term volatility forecasts.This paper is organized as follows: section 2 discusses the methodology we employed. Section 3 provides statistical characteristics of the stock market realized volatility and internet search volume data. Section 4 discusses the empirical results of the linkage between fluctuations in investor attention and stock market volatility. Section 5 discussed the forecasting power of models. Section 6 of this paper summarizes the conclusions.t, which can be expressed as:rt,i is the ith intraday return for day t, which can be calculated as ln−ln, and M denotes the numbers of intraday returns, we employ RV in the empirical analysis which is based on 5 minute intraday returns.Realized volatility (RV) prompted by Andersen and Bollerslev  can be cRV+ captures the market upturn and RV− captures the market downturn, where RV+ and RV− can be computed as:Following Barndorff-Nielsen, Kinnebrock, and Shephard , we decoFollowing Da, Engelberg, and Gao , we consMVd denotes the total market value of stock d, N represent the number of stocks in SSE50 and N equals 50, t refers to the day t, and d on day t. For instance, the Baidu search volume of Stock “ZhongGuoPingAn (601318)” can be obtained from Baidu Index with the Chinese words “中国平安”.While t and the mean value of attention index between day t−1 and day t−20, which can be expressed as:We construct attention fluctuation as the difference between the attention index on day We employ a Heterogeneous Autoregressive (HAR) model to investigate the relationship between investor attention fluctuation and stock market volatility. Based on heterogeneous market hypothesis of Müller et al. . Corsi  used a lWhere The HAR model transforms a basic AR model by imposing distinct restrictions on the autoregressive coefficients of the AR model, facilitating easy estimation through OLS. The ease of estimation coupled with superior performance makes the HAR model a favored choice for forecasting realized volatility.We incorporate investor attention fluctuation into a HAR model specification to proceed the analysis, the proposed HAR-AF model is as follows in i denotes the measures are the daily and weekly and monthly averages, the weekly and monthly RV can be calculated as RVt,stock denotes stock market realized volatility, and AFSmall, AFMedium, AFLarge, AFMajor denote small, medium, large, and major investor attention fluctuations respectively, Superscript “+” and “-” denote the positive  and negative  attention fluctuation and RV respectively.Where We estimate https://cn.gtadata.com/), and Baidu Index data can be obtained from Baidu Index (https://index.baidu.com/).We focus on the Shanghai Stock Exchange 50 Index (SSE50), and Baidu Index data from 10 May 2017 to 30 December 2022, total 1375 trading days. Our choice of SSE50 as a proxy for the Chinese stock market stems from its composition of the top 50 stocks, distinguished by the highest trading volume and capitalization on the Shanghai Stock Exchange. The constituents of SSE50 typically adjust semi-annually. Representing China’s core assets, the SSE50 has been frequently employed in existing literature examining Chinese stock market volatility. Following Da , we adopThe full-sample results are shown in We can find that daily and weekly average volatility measures are highly significant in both models underscoring the persistence of market volatility. Column 3 reveals that small fluctuation component exerts a negative impact on market volatility whereas the other three components have positive impact on market volatility (with the impacts of the large and major fluctuation components being significant). This suggests that small fluctuations stabilize the stock market while medium, large, and major fluctuations increase market volatility in full sample. This phenomenon may attribute to investor herding behavior. People purchase stocks as market fervor markedly escalates, consequently leading to increased volatility.Concerning positive large investor attention fluctuation, the absolute value of its coefficient exhibits a growing trend showing that large positive investor attention exerts weak and insignificant effect on stock market volatility, and its effect grows strong and become significant in the latter 3 subsamples. As for major attention fluctuation, the positive component shows a significant positive impact on market volatility in all subsamples, this aligns with the findings that increased investor attention, especially during significant market events, can lead to heightened market reactions and volatility, aligns with the results of Ballinari, Audrino, and Sigrist ; Chen, TLarge positive attention fluctuations are pivotal in increasing Chinese stock market volatility post COVID-19 outbreak. Conversely, small negative attention fluctuations significantly stabilize volatility pre-COVID-19, but this effect dwindles in post the pandemic. An inverse relationship exists between market sensitivity to positive large attention fluctuation and that to negative small attention fluctuation.The COVID-19 pandemic likely exacerbated market sensitivity to investor attention, particularly as it pertains to pandemic-related news and global economic uncertainties, people trade more when the heat of the stock rise. The pandemic possibly triggered a shift in investor behavior, making investors less reactive to market noise and more responsive to substantial market-impacting events. This behavioral shift could be fueled by the pervasive uncertainty and the global economic impact induced by the pandemic.Motivated by Gong and Lin , we subsThe results presented in Tables Our analysis predominantly revolves around the SSE 50 Index, which encapsulates the performance of 50 major companies listed on the Shanghai Stock Exchange. While this index reflects a significant portion of the market capitalization and trading activity, it does not entirely represent the broader dynamics and the diverse range of stocks within the entire Chinese stock market.Forecasting accuracy is also an essential aspect. We employ Eqs We apply 3 common loss functions to do the first comparison, namely the MSE, MAE and QLIKE loss function  test proposed by Hansen, Lunde, and Nason . FollowiRVt represents the actual value of realized volatility. In MCS test, the results are bootstrapped 10000 times and a confidence level of 0.05 is used. The results of one-step ahead forecasting MCS test, five-step ahead forecasting MCS test, and twenty-two steps forecasting MCS test are presented in Tables Sign “—” indicates model is eliminated. MCS test results confirm the superiority of the model incorporating decomposed RV and decomposed attention fluctuation in the short-term and medium-term forecasting. As for long-term forecasting, the model ranks the first under MSE1, MSE2 and R2LOG which still shows attention fluctuation can demystify the characteristics of stock market volatility.This paper pioneers the examination of the relationship between Chinese stock market realized volatility and investor attention fluctuation through the lens of the HAR model. Our nuanced analysis across full samples and sub-samples reveals critical insights into the dynamics of investor attention and stock market volatility.Positive attention fluctuations exceeding 7% are pivotal in influencing Chinese stock market volatility after the outbreak of COVID-19. Conversely, small negative attention fluctuations significantly impact volatility before COVID-19, but this effect dwindles in post pandemic. Notably, an inverse relationship exists between market sensitivity to positive large attention fluctuation and that to negative small attention fluctuation. Our models, encapsulating decomposed Realized Volatility (RV) and decomposed attention fluctuation, demonstrate superior predictive accuracy in forecasting short-term and medium-term realized volatility, as validated by model confidence set tests.Comprehending this nuanced relationship between investor attention fluctuations and stock market volatility aids investors utilizing attention fluctuations to forecast stock returns and formulate investment strategies. Policymakers can implement more regulations and guidance to steer investors towards value investing and exploit big data for regulatory purposes to mitigate extreme stock market volatility, preventing transmission of financial risks across global markets , 32, proSubsequent research could extend the analysis to a more representative array of indices or individual stocks across different market segments to gain a more holistic understanding of the relationship between investor attention fluctuations and market volatility.Our exploration enriches the behavioral finance discourse, particularly the investor attention effect, and provides empirical evidence of the multifaceted relationship between stock market volatility and varying levels of investor attention fluctuation over different time periods. Additionally, the superior performance of models incorporating decomposed RV and decomposed attention fluctuation in volatility forecasting underscores the potential for advancing financial modeling and risk assessment practices.


Capsicum annuum was used to perform individual differentially expressed gene analysis and to construct gene co-expression networks for each stress condition. Subsequently, gene networks were reconstructed around transcription factors to identify critical factors involved in the stress responses, including the NLR gene family, previously implicated in resistance. The abiotic and biotic stress networks comprise 233 and 597 hubs respectively, with 10 and 89 NLRs. Each gene within the NLR groups in the network exhibited substantial expression to particular stresses. The integrated analysis strategy of the transcriptome network revealed potential key genes for complex environmental conditions. Together, this could provide important clues to uncover novel key factors using high-throughput transcriptome data in other species as well as plants.Environmental stresses significantly affect plant growth, development, and productivity. Therefore, a deeper understanding of the underlying stress responses at the molecular level is needed. In this study, to identify critical genetic factors associated with environmental stress responses, the entire 737.3 Gb clean RNA-seq dataset across abiotic, biotic stress, and phytohormone conditions in  Plants encounter various types of stress, including abiotic factors, such as drought, heat, and salinity, as well as biotic factors, such as pathogen attacks and insect infestations3. These stressors induce complex molecular responses in plants leading to changes in gene expression, signaling pathways, and physiological adaptations5. Multiple studies have been conducted to identify genes involved in responses to diverse environmental stressors and one of the primary approaches utilizes transcriptome data for large-scale data analysis.Environmental stress is a major challenge for plants as it affects their growth, development, and productivity6. Along with RNA-seq, differential expression gene (DEG) analysis is a powerful approach to highlight gene expression changes. DEG has been widely used to identify key genes involved in responses to different treatments7. Gene expression patterns can also be utilized to analyze gene-gene interactions and to identify key regulatory factors in pathways and gene clusters8.RNA-seq is one of the main methods used to generate transcriptome data and has been used successfully in numerous studies to discover key genetic factors9. In pepper, key resistance genes were identified successfully through GCN analysis, which proved to be effective for identifying core regulators among the entire pool of resistance genes10. However, research that analyzes complex stress-based networks to identify key genetic factors are lacking.DEG-based gene co-expression networks (GCNs) play crucial roles in understanding the complex regulatory mechanisms underlying biological systems. These networks capture the relationships among genes based on their expression patterns under various conditions and/or in various tissues. By analyzing the co-expression patterns of genes, researchers gain insights into functional modules, regulatory pathways, and disease mechanisms13. Through this analysis process, we identified key regulatory genes that form the hub of the network and demonstrated the efficiency of the analysis method by identifying defense response genes that are highly expressed in the stress environment15.In this study, we introduce a method to identify key genes using GCNs based on RNA-seq data. By integrating and reconstructing individual networks around specific functions, this approach builds on the foundation of conventional RNA-seq analysis by placing greater emphasis on GCN construction. To demonstrate the efficiency of this method, we analyzed RNA-seq data from the pepper under environmental stress conditionsThis study uses a research methodology that involves constructing a network from RNA-seq data to identify regulators associated with the desired phenotype. Genes identified through this process are expected to play important roles. The dataset used in this study included abiotic and biotic stressors as well as phytohormone conditions. The procedural framework is shown in Fig. We obtained abiotic (204.7 Gb), biotic (344.6 Gb), and phytohormone 188 Gb) datasets from the NCBI Sequence Read Archive database (Table 88 Gb datPhytophthora capsici (Pc)-treated group exhibited the highest number of differentially expressed genes (DEGs) with 13,838 genes identified, whereas the pepper mottle virus (PepMoV)-treated group had the fewest DEGs with 545 genes  gene group, which is a major part of the disease-resistance gene family Fig. .Similarly, in the biotic stress environment, genes were grouped into eight clusters. Cluster 2 had the highest number of genes and the most pronounced changes in gene expression in the presence of Pc. We also observed a strong expression of NLR genes in the presence of Pc Fig. . Based oThis study presents a novel and effective approach that utilizes high-throughput RNA-seq data to identify key genetic factors involved in plant responses to environmental stress. Environmental stressors pose significant challenges to plants by affecting their growth, development, and productivity. To overcome these challenges and develop stress-tolerant crops, a deeper understanding of the underlying molecular responses is essential.In this study, we used RNA-seq data to analyze gene expression changes in response to different environmental stressors, including abiotic stress, biotic stress, and phytohormone treatments. Stressors induce complex molecular responses in plants leading to changes in gene expression, signaling pathways, and physiological adaptations. We used three primary analytical approaches: (1) DEG analysis, (2) GCN construction, and (3) integrated network construction. DEG analysis is a powerful method to identify genes that change expression significantly under specific stress conditions. By comparing gene expression levels between stress-treated and control samples, a significant number of DEGs associated with each stress condition were identified.Integration of the DEG-based GCNs plays a crucial role in understanding the complex regulatory mechanisms underlying biological systems. These networks capture the relationships between genes based on their expression patterns under different conditions and/or in different tissues. By analyzing the co-expression patterns of genes, researchers can gain important insights into functional modules, regulatory pathways, and disease mechanisms. The results of this study demonstrated the successful construction of GCNs for each stress condition and highlighted the interplay between stress-responsive genes and potential regulatory factors. The networks provided valuable information on gene interactions and revealed key regulators in stress responses.10. In this study, using an integrated network construction approach, we successfully identified TFs and NLR genes that respond to each stress condition confirming the acquisition of these key factors in the pepper16. In previous reports, NLR genes were found to be responsive to abiotic and biotic stresses, and the results of the present study also showed strong changes in their expression19. This research method proves to be a valuable tool for obtaining robust candidate resistance genes because it can be used effectively to identify important resistance-related genes.In addition, the integrated network approach proved particularly valuable because it allowed abiotic and biotic stress networks to be merged and allowed the identification of key interacting genes regulated by stress-related TFs. Thus, a comprehensive view of stress-responsive gene networks was formed that led to the identification of core regulatory modules associated with stress responses. Network analysis has been used successfully in several studies to identify gene clusters of interest, for example, in the pepper, it was used to identify key genes within the RLP gene familyIn conclusion, this study presents a comprehensive and systematic approach to identifying key genetic factors associated with plant responses to environmental stress. The integration of high-throughput RNA-seq data, DEG analysis, and GCNs provides valuable insights into stress-responsive gene interactions and regulatory mechanisms. The results will contribute to our understanding of plant stress responses and have the potential to advance the development of stress-tolerant crops to meet the challenges posed by environmental stressors in agriculture.P. capsici and P. infestans), two viruses (PepMoV and TMV-P0), two bacteria (Xcv1 and Xag8ra), and four phytohormones  were compared to determine the response of different environments (Table 20), biotic stress , signal molecule (SRP26526024). The gene expression patterns used in this study were obtained from the information registered in the Gene Expression Omnibus (GEO) of the NCBI .This study was conducted to select genes that respond to abiotic and biotic stresses using stress response RNA-seq to validate the efficiency of the assay. Responses four abiotic stressors , two oomycete pathogens  would be excluded from subsequent analysis. For read alignment, HISAT230 was employed using default parameters. The reference genome assembly used was the Capsicum annuum 2.0 version which was downloaded from PGENOME (http://peppergenome.snu.ac.kr).In the read quality control step of this study, adapter trimming was performed using Cutadapt31 in R. The following criteria were used to identify differentially expressed genes (DEGs): genes with an adjusted p-value (FDR) less than 0.05 and a log2fold change (log2FC) greater than |2|. DEGs were analyzed further for their functional implications.Transcript abundance was quantified using fragments per kilobase of transcript per million mapped reads (FPKM). Quantification was performed manually. DEG analysis was performed using the DESeq2 package32. DGCA evaluates interactions between genes based on co-expression patterns and forms a network structure. The network was constructed based on gene pairs with a correlation of 0.9 or more, and the TFs and GOs of the nodes in each connection were classified10. The co-expression network generated by DGCA was visualized using Cytoscape33. We constructed an integrated network by focusing on the genes shared within individual stress networks. The network was then reconstructed around the TFs for core network analysis, and direct correlation genes were obtained around the hub gene for analysis.Based on the DEGs, we constructed a co-expression network using the DGCA R packagehttps://www.biobam.com/omicsbox/) was used to functionally annotate the identified DEGs. OmicsBox 2.0 matched DEGs with available databases to retrieve functional annotations based on sequence homology and other relevant information. To further explore the functional implications of the DEGs, enrichment analysis was conducted using the goseq package34 in R. The goseq package incorporates the gene length bias inherent in RNA-seq data and performs gene ontology (GO) enrichment analysis. We focused on the stress-related gene ontology (GO: 50896) category to investigate genes associated with stress responses and adaptation. We reconfigured the integrated networks by focusing on the abiotic and biotic stress-related GO terms. We then performed expression profiling based on genes associated with stress-related GO terms. Heatmap analysis was performed to evaluate gene expression within a network. The expression levels of each gene were assessed using Z-scores to determine differences between treatment groups. To normalize expression levels based on treatment conditions, average values were calculated for each time point. This normalization allowed for comparative analyses of gene expression patterns across different treatments. For visualization, we used the ComplexHeatmap R package35.In this study, OmicsBox 2.0 (