Biology subjects

Sowa, A.

Publications and source records attributed to Sowa, A..

4 recordsLinked to original sources

bioRxiv2026

The Hao-Fountain syndrome gene USP7 restricts neurotropic orthoflavivirus entry through cell intrinsic control of endosomal dynamics

Neurodevelopmental disorders are increasingly associated with immune phenotypes, including autoinflammation, immunodeficiency, and increased susceptibility to severe infection. To determine whether neurodevelopmental disorders-associated genes exert immune functions, we performed an arrayed siRNA screen targeting 28 genes with nonredundant cellular roles and assessed their effects on Zika virus (ZIKV) infection and innate immune pathways. We identified hits that intrinsically restrict ZIKV infection and modulate inflammatory pathways following infection. We further characterized the antiviral activity of the Hao-Fountain syndrome gene USP7, which potently restricts selected neurotropic orthoflaviviruses. USP7 inhibits ZIKV internalization before viral membrane fusion and genome release into the cytoplasm. Because USP7 plays a role in endosomal tubulation and recycling, we investigated whether endosomal recycling pathways restrict ZIKV infection. We identified the USP7-regulated E3 ubiquitin ligase TRIM27, as well as the recycling-associated Rab GTPases RAB11 and RAB35, as potent regulators of ZIKV infection. Infection assays using cell lines expressing pathogenic USP7 variants and primary fibroblasts from individuals with Hao-Fountain syndrome demonstrated that disease-associated USP7 mutations impair its antiviral activity and increase permissivity to ZIKV infection. These findings are consistent with recent case reports of unusually severe viral infection during early life in individuals with Hao-Fountain syndrome. Collectively, our study identifies endosomal recycling pathways as important intrinsic restriction mechanisms against neurotropic orthoflaviviruses and nominates pathogenic USP7 variation as a candidate inborn error of immunity.

microbiology↗

bioRxiv2026

Perfusion quality does not necessarily predict ultrastructural preservation after hyperosmotic brain perfusion

Perfusion fixation is widely used in neuroscience to prepare mammalian brain tissue for histological and ultrastructural analysis. Perfusion protocols are commonly assessed using macroscopic indicators such as gross appearance and neuroimaging, which assess the extent to which perfusate has been distributed throughout the brain. There is a critical need to determine to what extent these metrics can accurately predict high-quality ultrastructural preservation, particularly as new perfusion protocols are developed for connectomics. In this technical report, we describe evidence that these two measures can be decoupled by the addition of dehydrating agents to the perfusate solution. In three human brain donors and one canine brain donor perfused with a fixative solution containing 10% mannitol and 10% polyethylene glycol 35 kDa, macroscopic and radiological indicators of perfusion quality appeared adequate or favorable. However, electron microscopy revealed expanded extracellular space, shrunken cellular processes, and distorted cell membranes, consistent with an osmotic shock artifact resulting from severe hyperosmotic dehydration. Similar ultrastructural artifacts were observed in a canine brain donor perfused with 20% mannitol in 20% neutral buffered formalin without PEG. We compare these ultrastructural findings with findings from previously reported cases perfused with standard neutral buffered formalin without osmotic additives. These findings illustrate a risk of optimizing brain perfusion protocols designed to preserve neural circuitry based on macroscopic or radiological perfusion quality metrics alone, since these metrics can be satisfied while the ultrastructure is severely compromised.

neuroscience↗

bioRxiv2026

Aldehyde-based cryopreservation of whole brains

Long-term storage of aldehyde-fixed brain tissue is commonly performed in the fluid state. This has the potential to maintain morphology for many decades, but has been found to cause progressive loss of antigenicity over time for some biomolecules. While cryoprotection and subzero storage has been successfully used for brain tissue sections or blocks, methods for preserving whole brains using this approach have not been widely characterized. Here we present a protocol for the preservation of fixed whole brains using graded immersion cryoprotection and subzero temperature storage, which is one type of a more general approach that we refer to as aldehyde-based cryopreservation (ABC). Our method uses a gradual ramp-up of the osmotic concentration of cryoprotectants, leading to a final solution containing 50% (v/v) ethylene glycol and 30% (w/v) sucrose. We used CT imaging to track cryoprotectant penetration, finding that with the use of our protocol, approximately 10 months is required to reach equilibration throughout whole human brains. In our initial histological validation, we found that insufficient equilibration time prior to freezing led to apparent ice crystal artifacts seen on ultrastructural imaging of the white matter. After refining the protocol to allow adequate diffusion time, histologic data at both the light and electron microscopic levels showed preserved cellular architecture and ultrastructure after the process of cryoprotectant loading, freezer storage, and unloading. This protocol can be implemented using laboratory freezers or freezer rooms and provides a degree of resilience against freezer failures because the morphology of the fixed tissue is expected to remain preserved long-term in the fluid state even if rewarmed. Our approach may be valuable for laboratories seeking to enhance the long-term preservation of antigenicity in large brain tissue specimens for future research applications.

neuroscience↗

bioRxiv2025

Evaluating ultrastructural preservation quality in banked brain tissue

The ultrastructural analysis of postmortem brain tissue can provide important insights into cellular architecture and disease-related changes. For example, connectomics studies offer a powerful emerging approach for understanding neural circuit organization. However, electron microscopy (EM) data is difficult to interpret when the preservation quality is imperfect, which is common in brain banking and may render it unsuitable for certain research applications. One common issue is that EM images of postmortem brain tissue can have an expansion of regions that appear to be made up of extracellular space and/or degraded cellular material, which we call ambiguous interstitial zones. In this study, we report a method to assess whether EM images have ambiguous interstitial zone artifacts in a cohort of 10 postmortem brains with samples from each of the cortex and thalamus. Next, in matched samples from the contralateral hemisphere of the same brains, we evaluate the structural preservation quality of light microscopy images, including immunostaining for cytoskeletal proteins. Through this analysis, we show that on light microscopy, cell membrane morphology can be largely maintained, and neurite trajectory visualized over micrometer distances, even in specimens for which there are ambiguous interstitial zone artifacts on EM. Taken together, our analysis may assist in maximizing the usefulness of donated brain tissue by informing tissue selection and preparation protocols for various research goals.

neuroscience↗