National Institute of Biomedical Imaging and Bioengineering

Platelet aggregation at sites of vascular injury is essential for hemostasis. However, the mechanisms that prevent excessive clot growth are not fully understood. In the prevailing "core and shell" model, based largely on small vessel injury studies, a central core of highly activated platelets is surrounded by a limited signal intensity shell of less activated, minimally degranulated platelets. Recent reports, especially in mouse models of profuse bleeding, suggest thrombus architecture and platelet activation states are more heterogeneous than the binary core and shell model proposes. Here, we performed high-resolution morphometric mapping of individual platelet activation states in mouse jugular vein and femoral artery puncture wound thrombi, using serial block face scanning electron microscopy and wide-area transmission electron microscopy. Manually annotated images were analyzed at multiple time points, revealing initial, 1-minute, near-complete intermixing of platelet activation states with no distinct core of highly activated platelets. At 5 minutes, highly activated, degranulated platelets became concentrated along the interior surfaces of vaulted thrombus structures. At 20 minutes, platelet numbers decreased and distinct clustering of degranulated, highly activated platelets was observed within central portions of the intravascular platelet-rich crown, limiting their access to the circulation. Deletion of the α-granule vesicle-soluble N-ethylmaleimide-sensitive factor attachment protein receptor, vesicle associated membrane protein 8, increased both the frequency and clustering of highly activated platelets. Similar patterns were observed in femoral artery wounds. We conclude that thrombus organization is more complex than previously recognized and provide evidence that progressive structural changes help limit procoagulant surface exposure and thrombus growth during hemostasis after puncture wounding.

Tissue-level phase transitions are emerging as a crucial mechanism in tumour development and metastasis. When becoming invasive, epithelial tumours undergo a transition from a solid-like state to a more fluid-like one. Although the contributions of cell adhesions, traction forces and cell migration for such behaviour are known, the exact biophysical and molecular mechanisms controlling these transitions are not fully understood. Here we show that breast cancer cell fluidity is regulated by IRSp53, a protein linking plasma membranes to the cytoskeleton. In both two-dimensional monolayers and three-dimensional spheroids, the depletion of IRSp53 increases fluidity and active wetting of the substrate due to a decrease in intercellular friction and enhanced local cell rearrangements. Molecularly, IRSp53 interacts with the junctional protein Afadin to control global tensile state and active wetting, establishing these proteins as key regulators of epithelial collectives' viscosity in breast cancer tumouroids. In breast cancer patient samples, low IRSp53 expression levels and aberrant localization correlate with worse clinical outcomes. These findings support the broader relevance of IRSp53-regulated mechanics in epithelia and their potential prognostic value in cancer.

Previously, we showed that the genetic diversity of SARS-CoV-2 nucleocapsid (N) protein explores a wide range of biophysical properties facilitated by non-local impact of point mutations to its intrinsically disordered regions (Nguyen et al., 2024). This includes modulation of self-association, such as the creation of a de novo binding interface through the P13L mutation characteristic of Omicron variants. In the present work, we focus on the key function of N condensing viral RNA into ribonucleoprotein particles (RNPs) for viral assembly. Lacking high-resolution structural information, biochemical and biophysical approaches have revealed architectural principles of RNPs, which involve cooperative interactions of several protein-protein and protein-RNA interfaces, initiated through oligomerization of conserved transient helices in the central disordered linker of N. Here, we study the impact of defining N-protein mutations in variants of concern on RNP formation, using biophysical tools, a virus-like particle assay, and reverse genetics experiments. We find convergent evolution in repeated, independent introduction of amino acid substitutions strengthening existing binding interfaces, compensating for other substitutions that promote viral replication but decrease RNP stability. Furthermore, we show that the P13L mutation of Omicron variants enhances RNP assembly and increases viral fitness. Overall, our data reveal RNP complexes to be highly variable not only in sequence and conformations but also in thermodynamic and kinetic stability, with its pleomorphism affecting basic architectural principles. We hypothesize that the formation of polydisperse, fuzzy N-RNA clusters with multiple distributed weak binding interfaces optimizes reversible RNA condensation, while supporting host adaptation and allowing for a large sequence space to be explored.

Highly pathogenic avian influenza (HPAI) A(H5N1) clade 2.3.4.4b was first detected in birds in 2021 in the United States (U.S.). An ongoing outbreak in dairy cattle began in early 2024 involving genotype B3.13. At least 70 U.S. cases have been identified in humans with exposure to infected cattle, poultry, and wild birds. No human-to-human transmission has yet been documented. However, for diagnostic preparedness, we evaluated the ability of currently available influenza tests to detect 2024 U.S. H5N1 genotypes, B3.13 and D1.1, predominant in wild birds and poultry. Contrived nasal swab samples were prepared using live or inactivated virus and used to test 12 rapid antigen tests (lateral flow assays, [LFA]), including 10 commercially available influenza A and two H5-specific LFAs. Five point-of-care (POC) molecular assays were also tested. An inclusivity testing protocol utilizing a predetermined dilution series was used to evaluate each assay, enabling a head-to-head comparison of performances. All LFAs and POC molecular tests were able to detect bovine 2024 H5N1 genotype B3.13. Sensitivity for the POC molecular tests (heat-inactivated B3.13) ranged from 1.55 to 7.75 TCID50/swab. For 11/12 LFAs, 10 commercial influenza A, and an RUO H5 assay, the sensitivity (live B3.13) ranged from 78 to 1550 TCID50/swab. The 12th LFA detected 77,500 TCID50/mL transport media. Testing of four LFAs confirmed inclusivity for genotype D1.1. Available influenza tests can detect 2024 U.S. H5N1 strains in contrived samples with a wide range of analytical sensitivity. In the event of human-to-human transmission, clinical performance and optimal sample types would need to be established.IMPORTANCETo date, at least 70 human cases of highly pathogenic avian influenza A (HPAI) H5N1 have been reported. Human-to-human transmission has not yet been reported, but there is ongoing HPAI H5N1 spread within animal and bird populations. As part of pandemic preparedness, it is critical to know whether available tests capable of detecting seasonal influenza A can also detect HPAI H5N1. Our paper describes results from the first study to assess and compare the ability of 10 commercially available influenza A lateral flow assays (LFAs), two LFAs specific for H5 influenza, and five POC molecular tests from three manufacturers to detect the H5N1 virus circulating in U.S. cattle (genotype B3.13). We found that all tests detect HPAI H5N1 in contrived nasal swab specimens, with varying degrees of analytical sensitivity. We also tested a subset of LFAs (three 510k-cleared COVID/flu multiplex tests and an RUO H5-specific LFA) to confirm that they were able to detect a D1.1 genotype (the predominant genotype in wild birds and poultry) strain isolated from a human case of H5N1 in Washington State. Given the persistent circulation of HPAI H5N1 cases, it is critical to know that available tests can analytically detect H5N1. Clinical performance and optimal sample types should be evaluated if human-to-human transmission is observed.

The nucleus and uropod are the largest and most mechanically distinct structures in migrating amoeboid lymphocytes, including NK, B, and T cells. The biophysical properties of these structures may shape the ability of immune cells to navigate dense tissue microenvironments during immune surveillance. Using bead-spring and agent-based cell models, we explore the biomechanical contributions of the nucleus, uropod, septin-templated cortical rings, actomyosin cytoskeleton, and extracellular matrix obstacles to lymphocyte migration. Our results support a migration model in which, following cell-matrix collisions, septins mediate the formation of cortical rings that hydraulically seal cytoplasmic compartments on each side of the passing nucleus, generating a pressure difference that propels the nucleus forward. This hydraulically driven nuclear piston actively enhances migration through confined spaces. Concurrently, the uropod emerging from the peristaltic collapse of rear compartments stabilizes directional persistence and prevents T cell repolarization. We show that such polarity stabilization boosts immune surveillance efficiency. Together, these models redefine the nucleus as an active component of the migratory engine and the uropod as a locomotion stabilizer. Furthermore, the models offer a predictive framework towards engineering of immune cell motility in complex tissue microenvironments with broad implications for cancer immunotherapy, aging, and regenerative medicine.

The integration of ex vivo perfusion machines for human organs prior to transplantation has improved outcomes for recipients and increased organ availability while revealing novel avenues of investigation for translational medicine. However, these machines have limited availability for experimental modifications while presenting a significant upfront cost investment and prohibitive per-use cost to the research scientist. Furthermore, there is a significant need for improved pre-clinical models in biomedical research that allow for authentic interrogation of cellular processes in the multi-cellular organ setting. To provide access to similar technology for the greater research community, we constructed a cost-effective modular normothermic perfusion machine entirely from readily sourced parts run with user-modifiable Python code. Since the system is devoid of customized hardware, investigators can construct and augment the system as required for individualized research applications. Herein, we describe the machine set-up and perfusion process in detail, convey our experience with porcine and human organs, and provide early proof-of-concept experimental results. These studies demonstrate the potential to interrogate pathophysiology and complex cellular processes using intact human tissue.