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The role of keratinocyte subpopulations in the different phases of the viral cycle during HPV16 infection remains to be characterised. Here, single cell RNA sequencing of HPV16 infected and uninfected organoids identifies 12 distinct keratinocyte populations including an HPV-reprogrammed keratinocyte subpopulation that is linked to cancer.

Abstract Both endovascular repair (EVR) and open repair (OR) surgery of thoraco-abdominal aortic aneurysms cause spinal cord (SC) injury that can lead to paraparesis or paraplegia. It has been assumed that mechanisms responsible for SC damage after EVR are similar to those after OR. This pilot study compared the pathophysiology of SC injury after EVR versus OR using a newly developed EVR dog model. An increasing number of stents similar to those used in patients were inserted in the aorta of three dogs to ensure thoracic or thoracic plus lumbar coverage. The aorta of OR dogs was cross-clamped for 45 min. Behavior assessment demonstrated unique patterns of proprioceptive ataxia and evolving paraparesis in EVR versus irreversible paraplegia in OR. MRI showed posterior signal in lumbar SC after EVR versus central cord edema after OR. Histopathology showed white matter edema in L3–L5 localized to the dorsal column medial lemniscus area associated with loss of myelin basic protein but not neurons after EVR, versus massive neuronal loss in the gray matter in L3–L5 after OR. Metabolome analysis demonstrates a distinctive chemical fingerprint of cellular processes in both interventions. Our results call for the development of new therapeutics tailored to these distinct pathophysiologic findings.

Aortic aneurism open repair surgery can cause spinal cord (SC) injury with 5–15% of patients developing paraparesis or paraplegia. Using a mouse model of transient aortic cross-clamping (ACC), we have previously found that the expression of proinflammatory microRNA miR-155 increases in motoneurons (MNs) and endothelial cells (ECs) of ischemic SCs, and that global miR-155 deletion decreases the percentage of paraplegia by 37.4% at 48-h post-ACC. Here, we investigated the cell-specific contribution of miR-155 in choline acetyltransferase-positive (ChAT+) neurons (that include all MNs of the SC) and ECs to SC injury after ACC. Mice lacking miR-155 in ChAT+ neurons (MN-miR-155-KO mice) developed 24.6% less paraplegia than control mice at 48-h post-ACC. In contrast, mice lacking miR-155 in ECs (ECs-miR-155-KO mice) experienced the same percentage of paraplegia as control mice, despite presenting smaller central cord edema. Unexpectedly, mice overexpressing miR-155 in ChAT+ neurons were less likely than control mice to develop early paraplegia during the first day post-ACC, however they reached the same percentage of paraplegia at 48-h. In addition, all mice overexpressing miR-155 in ECs (ECs-miR-155-KI mice) were paraplegic at 48-h post-ACC. Altogether, our results suggest that miR-155 activity in ChAT+ neurons protects the SC against ischemic injury during the first day post-ACC before becoming deleterious during the second day, which indicates that early and late paraplegias arise from different molecular malfunctions. These results point to the need to develop specific protective therapeutics aimed at inhibiting both the early and late deleterious events after open repair surgery of aortic aneurisms.

Autophagy is a proposed route of amyloid-β (Aβ) clearance by microglia that is halted in Alzheimer’s Disease (AD), though mechanisms underlying this dysfunction remain elusive. Here, primary microglia from adult AD (5xFAD) mice were utilized to demonstrate that 5xFAD microglia fail to degrade Aβ and express low levels of autophagy cargo receptor NBR1. In 5xFAD mouse brains, we show for the first time that AD microglia express elevated levels of microRNA cluster Mirc1/Mir17-92a, which is known to downregulate autophagy proteins. By in situ hybridization in post-mortem AD human tissue sections, we observed that the Mirc1/Mir17-92a cluster member miR-17 is also elevated in human AD microglia, specifically in the vicinity of Aβ deposits, compared to non-disease controls. We show that NBR1 expression is negatively correlated with expression of miR-17 in human AD microglia via immunohistopathologic staining in human AD brain tissue sections. We demonstrate in healthy microglia that autophagy cargo receptor NBR1 is required for Aβ degradation. Inhibiting elevated miR-17 in 5xFAD mouse microglia improves Aβ degradation, autophagy, and NBR1 puncta formation in vitro and improves NBR1 expression in vivo. These findings offer a mechanism behind dysfunctional autophagy in AD microglia which may be useful for therapeutic interventions aiming to improve autophagy function in AD.

Diffuse large B-cell lymphoma (DLBCL) is the most common non- Hodgkin lymphoma and is characterized by a remarkable heterogeneity with diverse variants that can be identified histologically and molecularly. Large-scale gene expression profiling studies have identified the germinal center B-cell (GCB-) and activated B-cell (ABC-) subtypes. Standard chemo-immunotherapy remains standard front-line therapy, curing approximately two thirds of patients. Patients with refractory disease or those who relapse after salvage treatment have an overall poor prognosis highlighting the need for novel therapeutic strategies. Transducin b-like protein 1 (TBL1) is an exchange adaptor protein encoded by the TBL1X gene and known to function as a master regulator of the Wnt signaling pathway by binding to β-CATENIN and promoting its downstream transcriptional program. Here, we show that, unlike normal B cells, DLBCL cells express abundant levels of TBL1 and its overexpression correlates with poor clinical outcome regardless of DLBCL molecular subtype. Genetic deletion of TBL1 and pharmacological approach using tegavivint, a first-in-class small molecule targeting TBL1 (Iterion Therapeutics), promotes DLBCL cell death in vitro and in vivo. Through an integrated genomic, biochemical, and pharmacologic analyses, we characterized a novel, β-CATENIN independent, post-transcriptional oncogenic function of TBL1 in DLBCL where TBL1 modulates the stability of key oncogenic proteins such as PLK1, MYC, and the autophagy regulatory protein BECLIN-1 through its interaction with a SKP1-CUL1-F-box (SCF) protein supercomplex. Collectively, our data provide the rationale for targeting TBL1 as a novel therapeutic strategy in DLBCL.

Dengue is an arboviral disease caused by dengue virus (DENV), which is transmitted to humans by Aedes aegypti mosquitoes. Infection by DENV most commonly results in a mild flu-like illness; however, the disease has been increasingly associated with neurological symptomatology. This association draws attention to further investigations on the impact of DENV infection in the host’s central nervous system. Here, we analyzed brain samples of three fatal dengue cases that occurred in 2002 during an outbreak in Rio de Janeiro, Brazil. Brain tissues of these cases were marked by histopathological alterations, such as degenerated neurons, demyelination, hemorrhage, edema, and increased numbers of astrocytes and microglial cells. Samples were also characterized by lymphocytic infiltrates mainly composed of CD8 T cells. DENV replication was evidenced in neurons, microglia and endothelial cells through immunohistochemistry and in situ hybridization techniques. Pro-inflammatory cytokines, such as TNF-α and IFN-γ were detected in microglia, while endothelial cells were marked by the expression of RANTES/CCL5. Cytoplasmic HMGB1 and the production of nitric oxide were also found in neurons and microglial cells. This work highlights the possible participation of several local pro-inflammatory mediators in the establishment of dengue neuropathogenesis.

Bovine leukemia virus (BLV), a deltaretrovirus, causes B-cell leukemia/lymphoma in cattle and is prevalent in herds globally. A previous finding of antibodies against BLV in humans led us to examine the possibility of human infection with BLV. We focused on breast tissue because, in cattle, BLV DNA and protein have been found to be more abundant in mammary epithelium than in lymphocytes. In human breast tissue specimens, we identified BLV DNA by using nested liquid-phase PCR and DNA sequencing. Variations from the bovine reference sequence were infrequent and limited to base substitutions. In situ PCR and immunohistochemical testing localized BLV to the secretory epithelium of the breast. Our finding of BLV in human tissues indicates a risk for the acquisition and proliferation of this virus in humans. Further research is needed to determine whether BLV may play a direct role in human disease.

Lung cancer is the leading cause of cancer-related deaths worldwide. Lack of early detection and limited options for targeted therapies are both contributing factors to the dismal statistics observed in lung cancer. Thus, advances in both of these areas are likely to lead to improved outcomes. MicroRNAs (miRs or miRNAs) represent a class of non-coding RNAs that have the capacity for gene regulation and may serve as both diagnostic and prognostic biomarkers in lung cancer. Abnormal expression patterns for several miRNAs have been identified in lung cancers. Specifically, let-7 and miR-9 are deregulated in both lung cancers and other solid malignancies. In this paper, we construct a mathematical model that integrates let-7 and miR-9 expression into a signaling pathway to generate an in silico model for the process of epithelial mesenchymal transition (EMT). Simulations of the model demonstrate that EGFR and Ras mutations in non-small cell lung cancers (NSCLC), which lead to the process of EMT, result in miR-9 upregulation and let-7 suppression, and this process is somewhat robust against random input into miR-9 and more strongly robust against random input into let-7. We elected to validate our model in vitro by testing the effects of EGFR inhibition on downstream MYC, miR-9 and let-7a expression. Interestingly, in an EGFR mutated lung cancer cell line, treatment with an EGFR inhibitor (Gefitinib) resulted in a concentration specific reduction in c-MYC and miR-9 expression while not changing let-7a expression. Our mathematical model explains the signaling link among EGFR, MYC, and miR-9, but not let-7. However, very little is presently known about factors that regulate let-7. It is quite possible that when such regulating factors become known and integrated into our model, they will further support our mathematical model.

MicroRNAs (miRNAs), single-stranded non-coding RNAs, influence myriad biological processes that can contribute to cancer. Although tumor-suppressive and oncogenic functions have been characterized for some miRNAs, the majority of microRNAs have not been investigated for their ability to promote and modulate tumorigenesis. Here, we established that the miR-191/425 cluster is transcriptionally dependent on the host gene, DALRD3, and that the hormone 17β-estradiol (estrogen or E2) controls expression of both miR-191/425 and DALRD3. MiR-191/425 locus characterization revealed that the recruitment of estrogen receptor α (ERα) to the regulatory region of the miR-191/425-DALRD3 unit resulted in the accumulation of miR-191 and miR-425 and subsequent decrease in DALRD3 expression levels. We demonstrated that miR-191 protects ERα positive breast cancer cells from hormone starvation-induced apoptosis through the suppression of tumor-suppressor EGR1. Furthermore, enforced expression of the miR-191/425 cluster in aggressive breast cancer cells altered global gene expression profiles and enabled us to identify important tumor promoting genes, including SATB1, CCND2, and FSCN1, as targets of miR-191 and miR-425. Finally, in vitro and in vivo experiments demonstrated that miR-191 and miR-425 reduced proliferation, impaired tumorigenesis and metastasis, and increased expression of epithelial markers in aggressive breast cancer cells. Our data provide compelling evidence for the transcriptional regulation of the miR-191/425 cluster and for its context-specific biological determinants in breast cancers. Importantly, we demonstrated that the miR-191/425 cluster, by reducing the expression of an extensive network of genes, has a fundamental impact on cancer initiation and progression of breast cancer cells.