Sequestration of Plasmodium falciparum-infected erythrocytes (IE) in the microvasculature is a major virulence determinant. While the sequestration of mature stage parasites (trophozoite and schizonts) to vascular endothelium is well established, the conditions that promote ring-stage IE sequestration are less understood. Here, we observed in ring-stage parasites that febrile exposure increased transcript levels of several exported parasite genes involved in the trafficking of the P. falciparum erythrocyte membrane protein 1 (PfEMP1) ligand responsible for adherence to the endothelium of blood vessels. Furthermore, it accelerated PfEMP1 surface display in ring-stage IEs, leading to a 2-fold increase in their binding in a perfusable 3D human brain microvessel model. Additionally, we observed that parasite exposure enhances the binding of uninfected erythrocytes in 3D brain microvessels. These findings suggest a complex interplay between fever and parasite biomass in the pathogenesis of cerebral malaria.

Bioengineered microvessels offer a controllable human system over cellular composition, vessel architecture, flow, and circulating components for studying vascular diseases. This protocol details the use of 3D brain microvessels to model in vivo-like parasite binding, maturation, and induced vascular inflammation during cerebral malaria progression. We outline steps for studying microvascular inflammation through immunofluorescence, ultrastructural imaging, RNA sequencing, and functional assays for permeability and leukocyte recruitment. This protocol can be adapted to investigate vascular dysfunction and blood-endothelium interactions in other diseases. For complete details on the use and execution of this protocol, please refer to Howard et al.1.

Disruption of the brain endothelial barrier is a hallmark of traumatic brain injury (TBI), and contributes to cerebral edema, coagulopathy, and delayed neurological deficits, yet there are no mechanism-guided therapies that directly stabilize human brain vessels. Here we used a three-dimensional (3D) human brain endothelial vessel platform with quantifiable macromolecular barrier function to assess responses to thrombin and plasma from patients with TBI. This endothelial-focused 3D system isolates the central cellular regulator of blood-brain barrier function and enables mechanistic analysis of endothelial signaling and drug responses as a foundation for personalized vascular therapeutics. By integrating this platform with machine learning-guided kinase analysis, we identified kinase pathways associated with barrier stabilization versus disruption and nominated inhibitors that reversed thrombin- and TBI plasma-induced leakage as well as compounds that exacerbated endothelial loss. This work establishes an important first step toward precision vascular therapeutics in TBI, by linking patient-derived plasma phenotypes to specific endothelial barrier responses and pharmacologically tractable kinase signaling, and it provides a generalizable framework for mechanistic drug discovery across neurovascular disorders. SIGNIFICANCE STATEMENT:Blood-brain barrier (BBB) disruption drives cerebral edema, coagulopathy, ischemia, and neurological deficits after TBI. Targeting kinases as therapeutics is promising but challenged by complex inhibitor profiles and patient variability. We present a machine learning-guided platform built on 3D perfusable human brain endothelial-centric microvessels to systematically screen and select kinase inhibitors that reverse TBI-induced endothelial barrier breakdown. By pinpointing key kinase and gene regulators and demonstrating restoration of endothelial barrier integrity in patient-derived plasma models, our approach provides a transformative strategy for stabilizing the endothelial barrier after TBI and offers a versatile framework for therapeutic discovery across neurovascular disorders.

Kinases are key players in endothelial barrier regulation, yet their temporal function and regulatory phosphosignaling networks are incompletely understood. We developed a novel methodology, Temporally REsolved KInase Network Generation (TREKING), which combines a 28-kinase inhibitor screen with machine learning and network reconstruction to build time-resolved, functional phosphosignaling networks. We demonstrated the utility of TREKING for identifying pathways mediating barrier integrity after activation by thrombin with or without TNF preconditioning in brain endothelial cells. TREKING predicted over 100 kinases involved in barrier regulation and discerned complex condition-specific pathways. For instance, the MAPK-activated protein kinase 2 (MAPKAPK2/MK2) had early barrier-weakening activity in both inflammatory conditions but late barrier-strengthening activity exclusively with thrombin alone. Using temporal Western blotting, we confirmed that MAPKAPK2/MK2 was differentially phosphorylated under the two inflammatory conditions. We further showed with lentivirus-mediated knockdown of MAPK14/p38α and drug targeting the MAPK14/p38α-MAPKAPK2/MK2 complex that a MAP3K20/ZAK-MAPK14/p38α axis controlled the late activation of MAPKAPK2/MK2 in the thrombin-alone condition. Beyond the MAPKAPK2/MK2 switch, TREKING predicts extensive interconnected networks that control endothelial barrier dynamics.

Summary During their northward migration, Red Knots Calidris canutus rufa stop at the Lagoa do Peixe National Park in the extreme south of Brazil to build up fat reserves for their journey to their Canadian breeding grounds. We tracked five Red Knots with PinPoint Argos-75 GPS transmitters to investigate differences in migration strategies from this stopover. Tracked birds used two different routes: the Central Brazil route and the Brazilian Atlantic Coast route. One bird flew 8,300 km straight from Lagoa do Peixe to the Delaware Bay (USA). Another bird stopped in Maranhão (north-east Brazil) and a third one used a yet unknown environment for the species, the mouth of the Amazon River at Baía Santa Rosa, Brazil. These two birds made short flights, covering stretches of 1,600 km to 3,600 km between stop-overs, where they stayed from 4 to 18 days. Our study highlights the occurrence of intrapopulation variation in migratory strategies and reveals the connectivity of environments that are essential for the viability of rufa Red Knot populations.

A hallmark of cerebral malaria is sequestration of Plasmodium falciparum-infected erythrocytes (IEs) in the brain microcirculation. Antibodies contribute to malaria immunity, but it remains unclear whether functional antibodies targeting parasite-expressed ligand can block cytoadhesion in the brain. Here, we screened the plasma of older children and young adults in Malawi to characterize the antibody response against the P. falciparum-IE surface and used a bioengineered 3-dimensional (3D) human brain microvessel model incorporating variable flow dynamics to measure adhesion-blocking responses. We found a strong correlation between surface antibody reactivity by flow cytometry and reduced P. falciparum-IE binding in 3D microvessels. Moreover, there was a threshold of surface antibody reactivity necessary to achieve robust inhibitory activity. Our findings provide evidence of the acquisition of adhesion-blocking antibodies against cerebral binding variants in people exposed to stable P. falciparum transmission and suggest the quality of the inhibitory response can be influenced by flow dynamics.

FLAT(n) is a tandem mass spectrometric techniquethatcan be used to rapidly generate spectral information applicable forstructural elucidation of lipids like lipid A from Gram-negative bacterialspecies from a single bacterial colony. In this study, we extend thescope and capability of FLAT(n) by tandem MS fragmentationof lithium-adducted molecular lipid A anions and fragments (FLAT(n-Li)) that provides additional structural and diagnosticdata from FLAT(n) samples allowing for the discriminationof terminal phosphate modifications in a variety of pathogenic andenvironmental species. Using FLAT(n-Li), we elucidatedthe lipid A structure from several bacterial species, including novelstructures from arctic bacterioplankton of the Duganella and Massilia genera that favor 4-amino-4-deoxy-l-arabinopyranose(Ara4N) modification at the 1-phosphate position and that demonstratedouble glycosylation with Ara4N at the 1 and 4 ' phosphate positionssimultaneously. The structures characterized in this work demonstratethat some environmental psychrophilic species make extensive use ofthis structural lipid A modification previously characterized as apathogenic adaptation and the structural basis of resistance to cationicantimicrobial peptides. This observation extends the role of phosphatemodification(s) in environmental species adaptation and suggests thatAra4N modification can functionally replace the positive charge ofthe phosphoethanolamine modification that is more typically foundattached to the 1-phosphate position of modified lipid A.

Sequestration of Plasmodium falciparum-infected erythrocytes (IEs) in the brain microcirculation is a hallmark of cerebral malaria (CM), which leads to endothelial activation, brain swelling, and death. Here, we probed CM inflammation in a perfusable 3D human brain microvessel model. 3D brain microvessels supported in vivo-like capacities for parasite binding and maturation in situ, leading to a distinct inflammatory response from the pro-inflammatory cytokine tumor necrosis factor a (TNF-a). By combining transcriptional analysis, imaging, and leukocyte perfusion, we showed that whereas TNF-a promotes a reversible inflammatory phenotype with widespread leukocyte recruitment, parasites induce unique stress response pathways and cause localized cell adhesivity changes, focal endothelial disruptions, and apoptosis. Furthermore, parasites modified the temporal kinetics of the TNF transcriptional response, suggesting augmented inflammatory damage with the two sequential stimuli. Our findings offer mechanistic insights into CM biology in a 3D brain microvessel mimetic platform and suggest that multiple events intersect to promote brain barrier inflammation in CM.

Many macro- and mesotidal estuaries are characterized by Turbidity Maxima Zones (TMZs), regions with suspended solid concentrations that are much higher than those found throughout the rest of the estuary. Such regions are located near the upriver limit of salt intrusion and their position and extent are modulated and driven by tidal oscillations, especially in estuaries where tidal forcing is large. Hence, pronounced TMZs are not typically expected in micro-tidal estuaries. Field experiments were carried out in the microtidal estuary of the Misa River (northeast coast of Italy) with the aim to analyze riverine-coastal ocean interactions during different climatic conditions, freshwater discharge and tidal forcing. The goal was also that of identifying factors and episodic conditions that could lead to the evolution of ephemeral TMZs in this microtidal estuarine system. Observational results, combined to a flocculation model suite, describe the hydrodynamics, morphological bed evolution, water chemistry and floc dynamics within the estuary during wintertime quiescent and stormy periods. Pronounced TMZs with different location and extent were observed during two storms with different intensities, when enhanced freshwater discharge, wave action and tidal oscillation generated significant stratification of the lower estuarine water column. Higher turbidity values were observed throughout the TMZ during the smaller/weaker storm, while stronger surface mixing during the stronger storm led to greater dispersion of the (re-)suspended particulate load throughout the upper water column, providing a less pronounced TMZ along the bed of the lower estuary. Observations in the Misa River, potentially valid for other microtidal estuaries, show that: 1) episodic storm conditions that significantly increase freshwater discharge can lead to the evolution of an ephemeral TMZ that is modulated, but not controlled, by tidal oscillations and surface mixing conditions; 2) ephemeral TMZ localization, intensity, and extent during episodic storm events is a function of storm intensity; 3) moderately enhanced freshwater flow during an episodic storm event promotes a high degree of stratification, allowing for the formation of large flocs with great settling rates, leading to a pronounced TMZ forming downriver of the landward limit of seawater intrusion; whereas higher freshwater flows during stronger storm events lead to less stratification, greater bottom turbulence and potential TMZ suppression near the riverbed, with shear conditions promoting smaller flocs with lower settling and a greater potential for suspended particulate export from the lower estuary to coastal waters.

Lay Summary center dot The eastern and western subspecies of Willets (Tringa semipalmata) are both declining and have little overlap in breeding and nonbreeding ranges. center dot Tracking and banding data show that Western Willets from Canada and the western United States wintered in California and Central America. Eastern Willets from the Atlantic Coast wintered in northern South America and those from the Gulf Coast wintered on the Pacific Coasts of Central America and Ecuador. center dot Both subspecies are threatened by habitat loss from climate change and development and the eastern subspecies has additional threats from hunting. center dot Strong migratory connectivity estimates between and within subspecies verify that subspecies-specific management actions are needed and indicate that population-specific actions are needed as well. center dot More information is needed on the migration of individuals in the center of the species range and where and when the two subspecies overlap to better understand where the two subspecies are relying on the same habitats and encountering the same threats. By combining all available banding and tracking data, we found that Willets (Tringa semipalmata) have a strong migratory connectivity between breeding and nonbreeding locations at the range-wide and subspecies levels, exposing two subspecies to varying threats such as hunting for the eastern subspecies (Tringa semipalmata semipalmata) and climatically-altered coastal habitats for both subspecies. We found that western Willets (Tringa semipalmata inornata) primarily used nonbreeding habitats along the Pacific Coast of the United States, although their reported nonbreeding range extends to the US Atlantic and Gulf Coasts and the Pacific Coast of Central and South America. Eastern Willets wintered in Central and South America, which covers much of the subspecies' known nonbreeding range. By quantifying migratory connectivity within and between two subspecies, we could suggest subspecies-specific threats and potential limiting factors in the breeding and nonbreeding periods of the annual cycle of a declining migratory shorebird. Effective management of the species will likely require a range of conservation strategies across the diverse nonbreeding regions the two subspecies occupy within the United States, Central America, and South America. However, more data are needed from Willets breeding in mid-continental North America to understand the complete extent of overlap of the two subspecies throughout the annual cycle. The strong migratory connectivity documented here highlights the need to manage Willets by subspecies and protect a diversity of breeding and nonbreeding habitats, which will benefit the conservation of other shorebird species that overlap with Willets throughout the annual cycle.

Cytoadhesion of Plasmodium falciparum-infected red blood cells is a virulence determinant associated with microvascular obstruction and organ complications. The gastrointestinal tract is a major site of sequestration in fatal cerebral malaria cases and kidney complications are common in severe malaria, but parasite interactions with these microvascular sites are poorly characterized. To study parasite tropism for different microvascular sites, we investigated binding of parasite lines to primary human microvascular endothelial cells from intestine (HIMEC) and peritubular kidney (HKMEC) sites. Of the three major host receptors for P. falciparum, CD36 had low or negligible expression; endothelial protein C receptor (EPCR) had the broadest constitutive expression; and intercellular adhesion molecule 1 (ICAM-1) was weakly expressed on resting cells and was strongly upregulated by TNF-α on primary endothelial cells from the brain, intestine, and peritubular kidney sites. By studying parasite lines expressing var genes linked to severe malaria, we provide evidence that both the DC8 and Group A EPCR-binding subsets of the P. falciparum erythrocyte membrane protein 1 (PfEMP1) family encodes binding affinity for brain, intestinal, and peritubular kidney endothelial cells, and that DC8 parasite adhesion was partially dependent on EPCR. Collectively, these findings raise the possibility of a brain-gut-kidney binding axis contributing to multi-organ complications in severe malaria.

Many species of shorebirds migrate long distances from their overwintering grounds in the southern hemisphere to breeding grounds in the northern hemisphere. The coastal intertidal zone, consisting of sand and mud flats exposed at low tide and covered at high tide, is heavily used as a migratory stopover or overwintering habitat. Understanding the spatial distribution of sediment types at these stopover sites is a critical step for understanding habitat use by shorebird species. Due to their importance as overwintering and stopover habitat for the imperiled western Atlantic subpopulation of the shorebird, the red knot (Calidris canutus rufa), as well as other migratory shorebirds, the northern coast of Brazil between Pará and Maranhão, and Bahía Lomas in northern Tierra del Fuego, Chile, were selected for further investigation as to the applicability of remotely sensed characterization of the intertidal flat habitats. Examination of the Landsat 8 multispectral reflectance and Sentinel-1 SAR backscatter reveals that sand and mud represent endmembers at opposite ends of a continuous gradient in feature space. While remotely sensed data can be used to discriminate between mud and sand intertidal types, the spectral relationships varied between the two very different geographic locations. The inclusion of both multispectral and radar sensing imagery can lead to important insights about the physical properties of the sediment that would be omitted by using one data source alone. Spectral unmixing techniques in Google Earth Engine were used to map the intertidal zone into general sediment classes spanning the gradient (i.e., mud, sandy mud, muddy sand, and sand). Comparison of the mapped outputs with field reference data suggests that mapping of mud- vs. sand-dominated areas can be accomplished with reasonable accuracy (overall accuracy of 75%).

The Malaria Evolution in South Asia (MESA) International Center for Excellence in Malaria Research (ICEMR) was established by the US National Institutes of Health (US NIH) as one of 10 malaria research centers in endemic countries. In 10 years of hospital-based and field-based work in India, the MESA-ICEMR has documented the changing epidemiology and transmission of malaria in four different parts of India. Malaria Evolution in South Asia-ICEMR activities, in collaboration with Indian partners, are carried out in the broad thematic areas of malaria case surveillance, vector biology and transmission, antimalarial resistance, pathogenesis, and host response. The program integrates insights from surveillance and field studies with novel basic science studies. This is a two-pronged approach determining the biology behind the disease patterns seen in the field, and generating new relevant biological questions about malaria to be tested in the field. Malaria Evolution in South Asia-ICEMR activities inform local and international stakeholders on the current status of malaria transmission in select parts of South Asia including updates on regional vectors of transmission of local parasites. The community surveys and new laboratory tools help monitor ongoing efforts to control and eliminate malaria in key regions of South Asia including the state of evolving antimalarial resistance in different parts of India, new host biomarkers of recent infection, and molecular markers of pathogenesis from uncomplicated and severe malaria.

Knowledge of the geographic linking of individuals or populations between different annual life cycle stages is essential for effective conservation decision making. The Willet (Tringa semipalmata) is composed of two distinct subspecies that are separated by breeding habitat in North America, with eastern Willets breeding in estuarine marshes along the Atlantic and Gulf Coasts and western Willets breeding in wet grasslands and prairies in the interior west of North America. We studied the migratory paths and wintering locations of Texas Gulf Coast breeding eastern Willets from 2015 to 2019 using light-level geolocators. Data analysis from 9 retrieved geolocators indicated that all birds departed Texas 5-26 July, made a 2-5-day flight, and arrived on the wintering grounds 8-30 July. All birds wintered on the Pacific coasts of Central and South America. In spring, the birds departed the wintering location 9-24 March, made a 3-4-day flight and arrived on the breeding grounds 12-27 March. These results are in contrast to previous work which showed that Atlantic breeding eastern Willets overwintered on the Atlantic coast in northern South America. This work has implications for shorebird conservation planning, which currently considers all Willets overwintering on the Pacific coast to be western Willets.

Cytoadhesion of Plasmodium falciparum-infected erythrocytes (IEs) to the endothelial lining of blood vessels protects parasites from splenic destruction, but also leads to detrimental inflammation and vessel occlusion. Surface display of the P. falciparum erythrocyte membrane protein 1 (PfEMP1) adhesion ligands exposes them to host antibodies and serum proteins. PfEMP1 are important targets of acquired immunity to malaria, and through evolution, the protein family has expanded and diversified to bind a select set of host receptors through antigenically diversified receptor-binding domains. Here, we show that complement component 1s (C1s) in serum cleaves PfEMP1 at semiconserved arginine motifs located at interdomain regions between the receptor-binding domains, rendering the IE incapable of binding the two main PfEMP1 receptors, CD36 and endothelial protein C receptor (EPCR). Bioinformatic analyses of PfEMP1 protein sequences from 15 P. falciparum genomes found the C1s motif was present in most PfEMP1 variants. Prediction of C1s cleavage and loss of binding to endothelial receptors was further corroborated by testing of several different parasite lines. These observations suggest that the parasites have maintained susceptibility for cleavage by the serine protease, C1s, and provides evidence for a complex relationship between the complement system and the P. falciparum cytoadhesion virulence determinant.

In this era of global environmental change and rapid regime shifts, managing core areas that species require to survive and persist is a grand challenge for conservation. Wildlife monitoring data are often limited or local in scale. The emerging ability to map and track spatial regimes (i.e., the spatial manifestation of state transitions) using advanced geospatial vegetation data has the potential to provide earlier warnings of habitat loss because many species of conservation concern strongly avoid spatial regime boundaries. Using 23 yr of data for the lek locations of Greater Prairie-Chicken (Tympanuchus cupido; GPC) in a remnant grassland ecosystem, we demonstrate how mapping changes in the boundaries between grassland and woodland spatial regimes provide a spatially explicit early warning signal for habitat loss for an iconic and vulnerable grassland-obligate known to be highly sensitive to woody plant encroachment. We tested whether a newly proposed metric for the quantification of spatial regimes captured well-known responses of GPC to woody plant expansion into grasslands. Resource selection functions showed that the grass:woody spatial regime boundary strength explained the probability of 80% of relative lek occurrence, and GPC strongly avoided grass:woody spatial regime boundaries at broad scales. Both findings are consistent with well-known expectations derived from GPC ecology. These results provide strong evidence for vegetation-derived delineations of spatial regimes to serve as generalized signals of early warning for state transitions that have major consequences to biodiversity conservation. Mapping spatial regime boundaries over time provided interpretable early warnings of habitat loss. Woody plant regimes displaced grassland regimes starting from the edges of the study area and constricting inward. Correspondingly, the relative probability of lek occurrence constricted in space. Similarly, the temporal trajectory of spatial regime boundary strength increased over time and moved closer to the observed limit of GPC lek site usage relative to grass:woody boundary strength. These novel spatial metrics allow managers to rapidly screen for early warning signals of spatial regime shifts and adapt management practices to defend and grow habitat cores at broad scales.

We know relatively little about the non-breeding period of most migratory birds. Decades of research on Swainson’s Thrush (Catharus ustulatus) has focused on their breeding ecology, whereas their non-breeding ecology is limited to broad patterns of distribution and migratory stop-over ecology. For the nearly 50 years, the US Breeding Bird Survey recorded declines of 0.7% per year for the Russet-backed Swainson’s Thrush (C. u. swainsonii) subspecies, which spends the non-breeding period in South America. However, there is an insufficient understanding of the constraints across their annual cycle to determine reasons for the sustained decline. In 2013 and 2014, we examined their little studied non-breeding period on the Andean slopes of Ecuador where prior experience showed that they were seasonally abundant. Here rapid deforestation threatens primary forest. We used point counts and radio telemetry to evaluate whether primary forest was used preferentially to recently regenerated second-growth forest. From point counts, 76% of detections and almost all captured individuals occurred in secondary forest. Of 86 birds captured in mist nets, 85 were males and 83 young of the year, indicating a highly skewed ratio in favor of young males. Radio telemetry demonstrated preference for secondary forest, especially for fruiting Cecropia spp. trees, with no apparent territorial behavior by those tracked and with short residency times. A marked decrease in density over the course of the field season suggests an itinerant population possibly tracking the ephemeral use of fruit resources. Alternatively, these temporal and spatial patterns could suggest that this population undergoes a mid-winter intratropical migration or at least landscape-level movements. However, we observed thrushes throughout our extended sampling period suggesting transiency with high turnover. Together, this study documents demographic separation during the non-breeding period with preference for of secondary forest and high transiency, important findings in informing management across the annual cycle.

Kinase inhibitors are promising drugs to stabilize the endothelial barrier following inflammatory damage. However, our limited knowledge of how kinase signaling activates barrier-restorative pathways and the complexity of multi-target drugs have hindered drug discovery and repurposing efforts. Here, we apply a kinase regression approach that exploits drug polypharmacology to investigate endothelial barrier regulation. A screen of 28 kinase inhibitors identified multiple inhibitors that promote endothelial barrier integrity and revealed divergent barrier phenotypes for BCR-ABL drugs. Target deconvolution predicted 50 barrier -regulating kinases from diverse kinase families. Using gene knockdowns, we identified kinases with a role in endothelial barrier regulation and dissected different mechanisms of action of barrier-protective kinase inhibitors. These results demonstrate the importance of polypharmacology in the endothelial barrier phenotype of kinase inhibitors and provide promising new leads for barrier-strengthening therapies.

Plasmodium falciparum pathogenesis is complex and intimately connected to vascular physiology. This is exemplified by cerebral malaria (CM), a neurovascular complication that accounts for most of the malaria deaths worldwide. P. falciparum sequestration in the brain microvasculature is a hallmark of CM and is not replicated in animal models. Numerous aspects of the disease are challenging to fully understand from clinical studies, such as parasite binding tropism or causal pathways in blood-brain barrier breakdown. Recent bioengineering approaches allow for the generation of 3D microvessels and organ-specific vasculature that provide precise control of vessel architecture and flow dynamics, and hold great promise for malaria research. Here, we discuss recent and future applications of bioengineered microvessels in malaria pathogenesis research.

Cerebral malaria (CM) affects children and adults, but brain swelling is more severe in children. To investigate features associated with brain swelling in malaria, we performed blood profiling and brain MRI in a cohort of pediatric and adult patients with CM in Rourkela, India, and compared them with an African pediatric CM cohort in Malawi. We determined that higher plasma Plasmodium falciparum histidine rich protein 2 (PfHRP2) levels and elevated var transcripts that encode for binding to endothelial protein C receptor (EPCR) were linked to CM at both sites. Machine learning models trained on the African pediatric cohort could classify brain swelling in Indian children CM cases but had weaker performance for adult classification, due to overall lower parasite var transcript levels in this age group and more severe thrombocytopenia in Rourkela adults. Subgrouping of patients with CM revealed higher parasite biomass linked to severe thrombocytopenia and higher Group A–EPCR var transcripts in mild thrombocytopenia. Overall, these findings provide evidence that higher parasite biomass and a subset of Group A–EPCR binding variants are common features in children and adult CM cases, despite age differences in brain swelling.