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In vitro, ACE2 translocates to the nucleus to induce SARS-CoV-2 replication. Here, using digital spatial profiling of lung tissues from SARS-CoV-2-infected golden Syrian hamsters, we show that a specific and selective peptide inhibitor of nuclear ACE2 (NACE2i) inhibits viral replication two days after SARS-CoV-2 infection. Moreover, the peptide also prevents inflammation and macrophage infiltration, and increases NK cell infiltration in bronchioles. NACE2i treatment increases the levels of the active histone mark, H3K27ac, restores host translation in infected hamster bronchiolar cells, and leads to an enrichment in methylated ACE2 in hamster bronchioles and lung macrophages, a signature associated with virus protection. In addition, ACE2 methylation is increased in myeloid cells from vaccinated patients and associated with reduced SARS-CoV-2 spike protein expression in monocytes from individuals who have recovered from infection. This protective epigenetic scarring of ACE2 is associated with a reduced latent viral reservoir in monocytes/macrophages and enhanced immune protection against SARS-CoV-2. Nuclear ACE2 may represent a therapeutic target independent of the variant and strain of viruses that use the ACE2 receptor for host cell entry. The entry receptor for SARS-CoV-2, ACE2, translocates to the nucleus following infection with this virus. Here, the authors demonstrate that a peptide targeting the ACE2 nuclear localization signal promotes its methylation and abrogates viral replication of SARS-CoV-2 in Syrian hamsters.

Ischemic stroke remains a leading cause of death and long-term disability, yet effective treatments that promote recovery beyond the acute phase are lacking. Neuregulin-1 (NRG-1) has shown potent neuroprotective and anti-inflammatory properties in preclinical stroke models, with evidence of enhanced neuronal regeneration when administered after injury. To investigate the spatial mechanisms underlying its neuroregenerative therapeutic effects, we examined brain proteomic responses to post-ischemic NRG-1 treatment in mice using NanoString Digital Spatial Profiling (DSP). Adult C57BL/6 mice were subjected to photothrombotic middle cerebral artery occlusion (MCAO) and treated with NRG-1β (5 μg/kg/day) or vehicle at 24- and 48-h post-stroke. Brains were collected at 3 days post-ischemia for spatial proteomic analysis of 68 neural proteins across the ischemic core, peri-infarct tissue, and peri-infarct normal tissue (PiNT). While NRG-1 did not significantly alter overall neuronal death, it markedly reshaped the neuroregenerative milieu, upregulating myelin basic protein (MBP) and synaptophysin and attenuating inflammatory mediators (SPP1, P2RX7, and CD39). NRG-1 also enhanced expression of autophagy and mitophagy markers (ULK1, LC3B, ATG5, PINK1, and Park7), suggesting restoration of cellular clearance and mitochondrial quality control. Pathway and network analyses revealed activation of neuroregeneration, autophagy, and lysosomal biogenesis pathways, while suppressing neuroinflammatory signaling. These findings demonstrate that delayed NRG-1 therapy, even when initiated 24 h after stroke, induces early molecular programs that prime an anti-inflammatory and neuroregenerative environment. The results support further development of NRG-1 as a clinically translatable, multimodal therapy for extending the post-stroke treatment window and promoting functional recovery.

Intra-tumor heterogeneity (ITH) is a major underlying cause of therapy resistance and disease recurrence, and is a read-out of tumor growth. Current genetic ITH analysis methods do not preserve spatial context and may not detect rare subclones. Here, we address these shortfalls by developing and validating BaseScope—a novel mutation-specific RNA in situ hybridization assay. We target common point mutations in the BRAF , KRAS and PIK3CA oncogenes in archival colorectal cancer samples to precisely map the spatial and morphological context of mutant subclones. Computational modeling suggests that subclones must arise sufficiently early, or carry a considerable fitness advantage, to form large or spatially disparate subclones. Examples of putative treatment-resistant cells isolated in small topographical areas are observed. The BaseScope assay represents a significant technical advance for in situ mutation detection that provides new insight into tumor evolution, and could have ramifications for selecting patients for treatment. Methods that analyze intra-tumor genetic heterogeneity often do not preserve the spatial context of tumor subclones. Here, the authors present BaseScope, a mutation-specific RNA in situ hybridization assay and spatially map colorectal cancer and adenoma KRAS , BRAF and PIK3CA driver gene mutant subclones.

Evidence has revealed that DDOST plays an important role in cancer development and progression. However, there are no reports on functions of DDOST in cervical tumorigenesis. Hence, we investigated the relationship of DDOST with prognosis, mutation, promoter methylation, immune cell infiltration, and drug sensitivity using bioinformatics techniques. Our results demonstrated that DDOST was significantly upregulated in a variety of tumor types and correlated with poor prognosis, including cervical cancer. Cox regression analysis dissected that high DDOST expression was associated with poor survival in cervical cancer patients. Immune infiltration analysis defined that DDOST was negatively correlated with CD8 T cells and NK cells. Strikingly, the sensitivity to multiple drugs was negatively correlated with the expression of DDOST. Therefore, our findings uncovered that DDOST could play an essential role in the tumor microenvironment and tumor immune regulation in cervical cancer, which indicated that DDOST could be a useful biomarker for prognosis and a potential therapeutic target for cancer treatment.

Background Latency remains a major obstacle to finding a cure for HIV despite the availability of antiretroviral therapy. Due to virus dormancy, limited biomarkers are available to identify latent HIV-infected cells. Profiling of individual HIV-infected cells is needed to explore potential latency biomarkers and to study the mechanisms of persistence that maintain the HIV reservoir. Methods Single cell spatial transcriptomic characterization using the CosMx Spatial Molecular Imager platform was conducted to analyze HIV-infected cells in formalin-fixed paraffin-embedded sections of splenic tissue surgically obtained from an HIV-infected humanized mouse model. Regulation of over a thousand human genes was quantified in both viremic and aviremic specimens. In addition, in situ hybridization and immunohistochemistry were performed in parallel to identify HIV viral RNA- and p24-containing cells, respectively. Finally, initial findings from CosMx gene profiling were confirmed by isolating RNA from CD4 + T cells obtained from a person living with HIV on antiretroviral therapy following either PMA/Ionomycin or DMSO treatment. RNA was quantified using qPCR for a panel of targeted human host genes. Results Supervised cell typing revealed that most of the HIV-infected cells in the mouse spleen sections were differentiated CD4 + T cells. A significantly higher number of infected cells, 2781 (1.61%) in comparison to 112 (0.06%), and total HIV transcripts per infected cell were observed in viremic samples compared to aviremic samples, respectively, which was consistent with the data obtained from ISH and IHC. Notably, the expression of 55 genes was different in infected cells within tissue from aviremic animals compared to viremic. In particular, both spleen tyrosine kinase ( SYK ) and CXCL17 , were expressed approximately 100-fold higher. This data was further evaluated against bulk RNA isolated from HIV-infected human primary CD4 + T cells. A nearly 6-fold higher expression of SYK mRNA was observed in DMSO-treated CD4 + T cells compared to those stimulated with PMA/Ionomycin. Conclusion This study found that the CosMx SMI platform is valuable for assessing HIV infection and providing insights into host biomarkers associated with HIV reservoirs. Higher relative expression of the SYK gene in aviremic-infected cells from the humanized mouse HIV model was consistent with levels found in CD4 + T cells of aviremic donors.

Objectives Severe Acute Respiratory Syndrome Coronavirus 2 (SARS‐CoV‐2) virus infection in pregnancy is associated with higher incidence of placental dysfunction, referred to by a few studies as a ‘preeclampsia‐like syndrome’. However, the mechanisms underpinning SARS‐CoV‐2‐induced placental malfunction are still unclear. Here, we investigated whether the transcriptional architecture of the placenta is altered in response to SARS‐CoV‐2 infection. Methods We utilised whole‐transcriptome, digital spatial profiling, to examine gene expression patterns in placental tissues from participants who contracted SARS‐CoV‐2 in the third trimester of their pregnancy (n = 7) and those collected prior to the start of the coronavirus disease 2019 (COVID‐19) pandemic (n = 9). Results Through comprehensive spatial transcriptomic analyses of the trophoblast and villous core stromal cell subpopulations in the placenta, we identified SARS‐CoV‐2 to promote signatures associated with hypoxia and placental dysfunction. Notably, genes associated with vasodilation (NOS3), oxidative stress (GDF15, CRH) and preeclampsia (FLT1, EGFR, KISS1, PAPPA2) were enriched with SARS‐CoV‐2. Pathways related to increased nutrient uptake, vascular tension, hypertension and inflammation were also enriched in SARS‐CoV‐2 samples compared to uninfected controls. Conclusions Our findings demonstrate the utility of spatially resolved transcriptomic analysis in defining the underlying pathogenic mechanisms of SARS‐CoV‐2 in pregnancy, particularly its role in placental dysfunction. Furthermore, this study highlights the significance of digital spatial profiling in mapping the intricate crosstalk between trophoblasts and villous core stromal cells, thus shedding light on pathways associated with placental dysfunction in pregnancies with SARS‐CoV‐2 infection. In this study, using spatial digital profiling transcriptomic approaches, we demonstrate that SARS‐CoV‐2 infection in pregnancy disrupts optimal placental function by altering the genomic architecture of trophoblasts and villous core stromal cells.

Studies of human cerebral cortex development are limited by difficulties in accessing and manipulating human neural tissue at specific development stages. We have derived human radial glia (hRG), which are responsible for most cerebral cortex neurogenesis, from human pluripotent stem cells. These hRG display the hallmark morphological, cellular, and molecular features of radial glia in vitro. They can be passaged and generate layer‐specific subtypes of cortical neurons in a temporal and passage‐dependent fashion. In later passages, they adopt a distinct progenitor phenotype that gives rise to cortical astrocytes and GABAergic interneurons. These hRG are also capable of following developmental cues to engraft, differentiate, migrate, and integrate into the embryonic mouse cortex when injected into E14 lateral ventricles. Moreover, hRG‐derived cells can be cryopreserved at specific stages and retain their stage‐specific phenotypes and competence when revived. Our study demonstrates that cultured hRG maintain a cell‐intrinsic clock that regulates the progressive generation of stage‐specific neuronal and glial subtypes. It also describes an easily accessible cell source for studying hRG lineage specification and progression and an on‐demand supply of specific cortical neuron subtypes and astrocytes. A protocol was successfully developed that generates developmentally accurate radial glia from human pluripotent stem cells. Such cultures can be passaged and frozen and demonstrate passage‐wise sequential generation of cortical neuron subtypes, cortical astrocytes, and interneurons. A late progenitor stage has also been characterized that has the capacity to generate large numbers of cortical astrocytes and dorsal‐derived cortical interneurons.

Astrogliosis with glial scar formation after damage to the nervous system is a major impediment to axonal regeneration and functional recovery. The present study examined the role of β1-integrin signaling in regulating astrocytic differentiation of neural stem cells. In the adult spinal cord β1-integrin is expressed predominantly in the ependymal region where ependymal stem cells (ESCs) reside. β1-integrin signaling suppressed astrocytic differentiation of both cultured ESCs and subventricular zone (SVZ) progenitor cells. Conditional knockout of β1-integrin enhanced astrogliogenesis both by cultured ESCs and by SVZ progenitor cells. Previous studies have shown that injection into the injured spinal cord of a self-assembling peptide amphiphile that displays an IKVAV epitope (IKVAV-PA) limits glial scar formation and enhances functional recovery. Here we find that injection of IKVAV-PA induced high levels of β1-integrin in ESCs in vivo, and that conditional knockout of β1-integrin abolished the astroglial suppressive effects of IKVAV-PA in vitro. Injection into an injured spinal cord of PAs expressing two other epitopes known to interact with β1-integrin, a Tenascin C epitope and the fibronectin epitope RGD, improved functional recovery comparable to the effects of IKVAV-PA. Finally we found that the effects of β1-integrin signaling on astrogliosis are mediated by integrin linked kinase (ILK). These observations demonstrate an important role for β1-integrin/ILK signaling in regulating astrogliosis from ESCs and suggest ILK as a potential target for limiting glial scar formation after nervous system injury.

An early substantial loss of basal forebrain cholinergic neurons (BFCNs) is a constant feature of Alzheimer’s disease (AD) and is associated with deficits in spatial learning and memory. Induced pluripotent stem cells (iPSCs) derived from patients with AD as well as from normal controls could be efficiently differentiated into neurons with characteristics of BFCNs. We used BFCNs derived from iPSCs to model sporadic AD with a focus on patients with ApoE3/E4 genotypes (AD-E3/E4). BFCNs derived from AD-E3/E4 patients showed typical AD biochemical features evidenced by increased Aβ42/Aβ40 ratios. AD-E3/E4 neurons also exhibited altered responses to treatment with γ-secretase inhibitors compared to control BFCNs or neurons derived from patients with familial AD. BFCNs from patients with AD-E3/E4 also exhibited increased vulnerability to glutamate-mediated cell death which correlated with increased intracellular free calcium upon glutamate exposure. The ability to generate BFCNs with an AD phenotype is a significant step both for understanding disease mechanisms and for facilitating screening for agents that promote synaptic integrity and neuronal survival.

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.