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The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is known to present with pulmonary and extra-pulmonary organ complications. In comparison with the 2009 pandemic (pH1N1), SARS-CoV-2 infection is likely to lead to more severe disease, with multi-organ effects, including cardiovascular disease. SARS-CoV-2 has been associated with acute and long-term cardiovascular disease, but the molecular changes govern this remain unknown.In this study, we investigated the landscape of cardiac tissues collected at rapid autopsy from SARS-CoV-2, pH1N1, and control patients using targeted spatial transcriptomics approaches. Although SARS-CoV-2 was not detected in cardiac tissue, host transcriptomics showed upregulation of genes associated with DNA damage and repair, heat shock, and M1-like macrophage infiltration in the cardiac tissues of COVID-19 patients. The DNA damage present in the SARS-CoV-2 patient samples, were further confirmed by γ−H2Ax immunohistochemistry. In comparison, pH1N1 showed upregulation of Interferon-stimulated genes (ISGs), in particular interferon and complement pathways, when compared with COVID-19 patients.These data demonstrate the emergence of distinct transcriptomic profiles in cardiac tissues of SARS-CoV-2 and pH1N1 influenza infection supporting the need for a greater understanding of the effects on extra-pulmonary organs, including the cardiovascular system of COVID-19 patients, to delineate the immunopathobiology of SARS-CoV-2 infection, and long term impact on health.

In recent years, pregnant people infected with the SARS-CoV-2 virus have shown a higher incidence of “preeclampsia-like syndrome”. Preeclampsia is a systematic syndrome that affects 5-8 % of pregnant people worldwide and is the leading cause of maternal mortality and morbidity. It is unclear what causes preeclampsia, and is characterised by placental dysfunction, leading to poor placental perfusion, maternal hypertension, proteinuria, thrombocytopenia, or neurological disturbances.In this study, we used whole-transcriptome, digital spatial profiling of placental tissues to analyse the expression of genes at the cellular level between placentae from pregnant participants who contracted SARS-CoV-2 in the third trimester of their pregnancy and those prior to the start of the pandemic. Our focused analysis of the trophoblast and villous core stromal cell populations revealed tissue-specific pathways enriched in the SARS-CoV-2 placentae that align with a pre-eclampsia signature. Most notably, we found enrichment of pathways involved in vascular tension, blood pressure, inflammation, and oxidative stress.This study illustrates how spatially resolved transcriptomic analysis of placental tissue can aid in understanding the underlying pathogenic mechanisms of SARS-CoV-2 in pregnancy that are thought to induce “preeclampsia-like syndrome”. Moreover, our study highlights the benefits of using digital spatial profiling to map the crosstalk between trophoblast and villous core stromal cells linked to pathways involved in “preeclampsia-like syndrome” presenting in pregnant people with SARS-CoV-2.

Bioengineering facets of the tumour microenvironment (TME) are essential in 3D tissue models to accurately recapitulate tumour progression. Stromal cells are key components of the TME and their incorporation into 3D biomimetic bioengineered tumour-stroma models is essential to be able to mimic the TME. By engineering tumouroids with distinct tumour and stromal compartments, it has been possible to identify how gene expression is altered by the presence of different stromal cells using spatial transcriptomics. Ameloblastoma is a benign epithelial tumour of the jawbone and in engineered multi-compartment tumouroids increased expression of oncogenes was found where osteoblasts (bone stroma) were present. Engineering a gingival fibroblast stroma resulted in increased matrix remodelling genes in the ameloblastoma tumour. This study provides evidence to show the stromal specific effect on tumour behaviour and illustrates the importance of engineering biologically relevant stroma for engineered tumour models. Our novel results show that an engineered fibroblast stroma causes the upregulation of matrix remodelling genes in ameloblastoma which directly correlates to measured invasion in the model. In contrast the presence of an osteoblast/bone stroma increases the expression of oncogenes by ameloblastoma cells.

Neuregulin-1 (NRG-1) has been shown to be neuroprotective and anti-inflammatory in rodent models following focal brain ischemia. However, the spatial transcriptional mechanisms involved in the effects of NRG-1 have not been investigated. In this study, we examined the spatial cellular and neuroinflammatory mechanisms employed by NRG-1 following stroke utilizing Nanostring Digital Spatial Profiling (DSP) technology. C57bl/6 mice were subjected to photothrombotic middle cerebral artery occlusion (MCAO). Animals were treated with NRG-1 (5ug/kg/day NRG-1 β) or vehicle beginning 24 hours after surgery, and then sacrificed at 3 days post-ischemia (dpi). Using GeoMX DSP technology, we examined the relative expression of 73 proteins in selected regions of interest (ROI) within the ischemic core, peri-infarct tissues, and peri-infarct normal tissue (PiNT) in controls and NRG-1 treated mice. FJB staining showed that delayed NRG-1 treatment did not significantly alter overall neuronal death, but it had profound effects on the neuroregenerative outgrowth environment. The ipsilateral core ROI demonstrated the most altered proteomic profile with 37 differentially regulated proteins related to autophagy and mitophagy, immune cell phagocytosis, and synaptic activity. NRG-1 upregulated synaptophysin by 2.10-fold while marginally rescuing neuronal proteins NeuN and MAP2. NRG-1 treatment induced the upregulation of ULK1, LC3B, ATG5, PINK1, and Park7 within the core region, suggesting successful autophagic flux restoration. NRG-1 treatment also increased Mertk, CD9, CSF1R/CD115, MHCII and Myelin Basic Protein (MBP). Phospho-Tau (S199) was increased 57-fold following stroke and further increased to 90-fold after NRG-1 treatment. The peri-infarct region showed increased astrocyte proteins along with BAG3, CD204/MSR1, phospho-Tau (S199) and Ki67, a marker of cell proliferation. Together, our findings showed that mechanisms of neuroprotection by NRG-1 following stroke occur in distinct spatial domains of the injured brain following ischemia. The data suggest that NRG-1 provides protection against further neuronal apoptosis and priming of an anti-inflammatory, neuroregenerative niche in a spatial manner, which could lead to novel treatment strategies for stroke.

The molecular underpinnings of organ dysfunction in acute COVID-19 and its potential long-term sequelae are under intense investigation. To shed light on these in the context of liver function, we performed single-nucleus RNA-seq and spatial transcriptomic profiling of livers from 17 COVID-19 decedents. We identified hepatocytes positive for SARS-CoV-2 RNA with an expression phenotype resembling infected lung epithelial cells. Integrated analysis and comparisons with healthy controls revealed extensive changes in the cellular composition and expression states in COVID-19 liver, reflecting hepatocellular injury, ductular reaction, pathologic vascular expansion, and fibrogenesis. We also observed Kupffer cell proliferation and erythrocyte progenitors for the first time in a human liver single-cell atlas, resembling similar responses in liver injury in mice and in sepsis, respectively. Despite the absence of a clinical acute liver injury phenotype, endothelial cell composition was dramatically impacted in COVID-19, concomitantly with extensive alterations and profibrogenic activation of reactive cholangiocytes and mesenchymal cells. Our atlas provides novel insights into liver physiology and pathology in COVID-19 and forms a foundational resource for its investigation and understanding.

Understanding the physiology and functions of the liver and cancer requires knowing transcriptional patterns driving biological activities within the functional structures of the tissue, especially the zonated features of the liver metabolic networks. Using the powerful and unique capabilities of GeoMx® Digital Spatial Profiler (DSP) with the Whole Transcriptome Atlas (WTA) panel to resolve functional units within FFPE tissues in situ, here we report the spatial analysis of whole transcriptomes across three micro-dissected zones (pericentral zone 3, intermediate zone 2 and periportal zone 1) of human normal liver and HCC. We also report the whole transcriptome expression data from Kupffer cells, portal traits and interlobular bile ducts from four normal liver samples and HCC. 500 - 3000 genes were detected from functional groups within each histological structure. In the liver functional units, 1000 - 2500 genes were detected in zone 1, 2 and 3 separately. By comparing the whole transcriptome profiles of zone 1 and zone 3, we have found 32 differentially expressed targets (fold change > 1.5, p value < 0.05) which showed a gradient expression pattern along the porto-central axis. The expression patterns of CYP1A2, CYP2E1, CYP3A4 and ALDOB matched well with their respective patterns of protein expression (Human Protein Atlas), recapitulating the well-studied distribution of functional activities along the porto-central axis. Moreover, by combining with Gene Set Enrichment Analysis (GSEA), we have found important pathways involved in metabolisms in either the pericentral area or the periportal area. Pathways including biological oxidations (CYP1A2, CYP2E1, CYP3A4, ADH1A, and ADH1B) and lipids metabolism (AKR1C1, AKR1C2, and SLCO1B3) showed high enrichment in zone 3 and decreased towards zone 1. In contrast, pathways including platelet degranulation (FGA, FGB, FGG), glucose metabolism (ALDOB and PCK1) and amino acids metabolism (HAL, SDS, NNMT, and GLS2) showed high enrichment in zone 1 and decreased towards zone 3. In conclusion, our WTA data has revealed clear metabolic zonation in the liver along the porto-central axis. GeoMx technology with WTA is a powerful tool to investigate the underlying mechanisms of liver metabolism, regeneration, and tissue structure. It can be further utilized to study the whole transcriptomic differences in normal and diseased tissue. FOR RESEARCH USE ONLY. Not for use in diagnostic procedures. Citation Format: Yan Liang, Nan Wang, Xia Li, Kathy Ton, Liang Zhang, Megan Vandenberg, Charlie Glaser, Zhiyong Ding, Joseph Beechem, Andy Nam, Yan Liang. Spatial whole transcriptome profiling of human normal liver and HCC uncovers unique insights into metabolic zonation. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4709.

Stroke is ranked as the fifth leading cause of death and the leading cause of adult disability in the USA. The progression of neuronal damage after stroke is recognized to be a complex integration of glia, neurons, and the surrounding extracellular matrix, therefore potential treatments must target the detrimental effects created by these interactions. In this study, we examined the spatial cellular and neuroinflammatory mechanisms occurring early after ischemic stroke utilizing Nanostring Digital Spatial Profiling (DSP) technology. Male C57bl/6 mice were subjected to photothrombotic middle cerebral artery occlusion (MCAO) and sacrificed at 3 days post-ischemia. Spatial distinction of the ipsilateral hemisphere was studied according to the regions of interest: the ischemic core, peri-infarct tissues, and peri-infarct normal tissue (PiNT) in comparison to the contralateral hemisphere. We demonstrated that the ipsilateral hemisphere initiates distinct spatial regulatory proteomic profiles with DSP technology that can be identified consistently with the immunohistochemical markers, FJB, GFAP, and Iba-1. The core border profile demonstrated an induction of neuronal death, apoptosis, autophagy, immunoreactivity, and early degenerative proteins. Most notably, the core border resulted in a decrease of the neuronal proteins Map2 and NeuN; an increase in the autophagy proteins BAG3 and CTSD; an increase in the microglial and peripheral immune invasion proteins Iba1, CD45, CD11b, and CD39; and an increase in the neurodegenerative proteins BACE1, APP, amyloid β 1–42, ApoE, and hyperphosphorylated tau protein S-199. The peri-infarct region demonstrated increased astrocytic, immunoreactivity, apoptotic, and neurodegenerative proteomic profiles, with an increase in BAG3, GFAP, and hyperphosphorylated tau protein S-199. The PiNT region displayed minimal changes compared to the contralateral cortex with only an increase in GFAP. In this study, we showed that mechanisms known to be associated with stroke, such as apoptosis and inflammation, occur in distinct spatial domains of the injured brain following ischemia. We also demonstrated the dysregulation of specific autophagic pathways that may lead to neurodegeneration in peri-infarct brain tissues. Taken together, these data suggest that identifying post-ischemic mechanisms occurring in a spatiotemporal manner may lead to more precise targets for successful therapeutic interventions to treat stroke.

Maize shoot development progresses from non-pigmented meristematic cells at the base of the leaf to expanded and non-dividing green cells of the leaf blade. This transition is accompanied by the conversion of promitochondria and proplastids to their mature forms and massive fragmentation of both mitochondrial DNA (mtDNA) and plastid DNA (ptDNA), collectively termed organellar DNA (orgDNA). We measured developmental changes in reactive oxygen species (ROS), which at high concentrations can lead to oxidative stress and DNA damage, as well as antioxidant agents and oxidative damage in orgDNA. Our plants were grown under normal, non-stressful conditions. Nonetheless, we found more oxidative damage in orgDNA from leaf than stalk tissues and higher levels of hydrogen peroxide, superoxide, and superoxide dismutase in leaf than stalk tissues and in light-grown compared to dark-grown leaves. In both mitochondria and plastids, activities of the antioxidant enzyme peroxidase were higher in stalk than in leaves and in dark-grown than light-grown leaves. In protoplasts, the amount of the small-molecule antioxidants, glutathione and ascorbic acid, and catalase activity were also higher in the stalk than in leaf tissue. The data suggest that the degree of oxidative stress in the organelles is lower in stalk than leaf and lower in dark than light growth conditions. We speculate that the damaged/fragmented orgDNA in leaves (but not the basal meristem) results from ROS signaling to the nucleus to stop delivering DNA repair proteins to mature organelles producing large amounts of ROS.