Probes for INS

About 50% of hospitalized coronavirus disease 2019 (COVID-19) patients with diabetes mellitus (DM) developed myocardial damage. The mechanisms of direct SARS-CoV-2 cardiomyocyte infection include viral invasion via ACE2-Spike glycoprotein-binding. In DM patients, the impact of glycation of ACE2 on cardiomyocyte invasion by SARS-CoV-2 can be of high importance. To evaluate the presence of SARS-CoV-2 in cardiomyocytes from heart autopsy of DM cases compared to Non-DM; to investigate the role of DM in SARS-COV-2 entry in cardiomyocytes. We evaluated consecutive autopsy cases, deceased for COVID-19, from Italy between Apr 30, 2020 and Jan 18, 2021. We evaluated SARS-CoV-2 in cardiomyocytes, expression of ACE2 (total and glycosylated form), and transmembrane protease serine protease-2 (TMPRSS2) protein. In order to study the role of diabetes on cardiomyocyte alterations, independently of COVID-19, we investigated ACE2, glycosylated ACE2, and TMPRSS2 proteins in cardiomyocytes from DM and Non-DM explanted-hearts. Finally, to investigate the effects of DM on ACE2 protein modification, an in vitro glycation study of recombinant human ACE2 (hACE2) was performed to evaluate the effects on binding to SARS-CoV-2 Spike protein. The authors included cardiac tissue from 97 autopsies. DM was diagnosed in 37 patients (38%). Fourth-seven out of 97 autopsies (48%) had SARS-CoV-2 RNA in cardiomyocytes. Thirty out of 37 DM autopsy cases (81%) and 17 out of 60 Non-DM autopsy cases (28%) had SARS-CoV-2 RNA in cardiomyocytes. Total ACE2, glycosylated ACE2, and TMPRSS2 protein expressions were higher in cardiomyocytes from autopsied and explanted hearts of DM than Non-DM. In vitro exposure of monomeric hACE2 to 120 mM glucose for 12 days led to non-enzymatic glycation of four lysine residues in the neck domain affecting the protein oligomerization. The upregulation of ACE2 expression (total and glycosylated forms) in DM cardiomyocytes, along with non-enzymatic glycation, could increase the susceptibility to COVID-19 infection in DM patients by favouring the cellular entry of SARS-CoV2.

Cardiac involvement in COVID-19 is associated with adverse outcome. However, it is unclear whether cell specific consequences are associated with cardiac SARS-CoV-2 infection. Therefore, we investigated heart tissue utilizing in situ hybridization, immunohistochemistry and RNA-sequencing in consecutive autopsy cases to quantify virus load and characterize cardiac involvement in COVID-19.In this study, 95 SARS-CoV-2-positive autopsy cases were included. A relevant SARS-CoV-2 virus load in the cardiac tissue was detected in 41/95 deceased (43%). MACE-RNA-sequencing was performed to identify molecular pathomechanisms caused by the infection of the heart. A signature matrix was generated based on the single-cell dataset "Heart Cell Atlas" and used for digital cytometry on the MACE-RNA-sequencing data. Thus, immune cell fractions were estimated and revealed no difference in immune cell numbers in cases with and without cardiac infection. This result was confirmed by quantitative immunohistological diagnosis.MACE-RNA-sequencing revealed 19 differentially expressed genes (DEGs) with a q-value <0.05 (e.g. up: IFI44L, IFT3, TRIM25; down: NPPB, MB, MYPN). The upregulated DEGs were linked to interferon pathways and originate predominantly from endothelial cells. In contrast, the downregulated DEGs originate predominately from cardiomyocytes. Immunofluorescent staining showed viral protein in cells positive for the endothelial marker ICAM1 but rarely in cardiomyocytes. The GO term analysis revealed that downregulated GO terms were linked to cardiomyocyte structure, whereas upregulated GO terms were linked to anti-virus immune response.This study reveals, that cardiac infection induced transcriptomic alterations mainly linked to immune response and destruction of cardiomyocytes. While endothelial cells are primarily targeted by the virus, we suggest cardiomyocyte-destruction by paracrine effects. Increased pro-inflammatory gene expression was detected in SARS-CoV-2-infected cardiac tissue but no increased SARS-CoV-2 associated immune cell infiltration was observed.Cardiac injury can be documented in COVID-19, regardless the direct cardiac virus infection and is known to be associated with outcome. However, the direct virus infection of the myocardium leads to transcriptomic alterations and might therefore additionally contribute to pathophysiological processes in COVID-19. Therefore, consequences of cardiac virus infection need to be investigated in future studies, since they might also contribute to long-term effects in case of survival.

We evaluated the potential of AGTR1, the principal receptor for angiotensin II (Ang II) and a member of the G protein-coupled receptor family, for targeted delivery of antisense oligonucleotides (ASOs) in cells and tissues with abundant AGTR1 expression. Ang II peptide ASO conjugates maintained robust AGTR1 signaling and receptor internalization when ASO was placed at the N-terminus of the peptide, but not at C-terminus. Conjugation of Ang II peptide improved ASO potency up to 12- to 17-fold in AGTR1-expressing cells. Additionally, evaluation of Ang II conjugates in cells lacking AGTR1 revealed no enhancement of ASO potency. Ang II peptide conjugation improves potency of ASO in mouse heart, adrenal, and adipose tissues. The data presented in this report add to a growing list of approaches for improving ASO potency in extrahepatic tissues.

Recent studies suggest onco-regulatory roles for two long non-coding RNAs (lncRNAs), MALAT1 and HOTAIR, in various malignancies; however, these lncRNAs have not been previously examined in neuroendocrine neoplasms (NENs) of gastroenteropancreatic origins (GEP-NENs). In this study, we evaluated the expressions and prognostic significance of MALAT1 and HOTAIR in 83 cases of GEP-NENs (60 grade 1, 17 grade 2, and 6 grade 3 tumors) diagnosed during the years 2005-2017. Expression levels of MALAT1 and HOTAIR were digitally quantitated in assembled tissue microarray slides labeled by chromogenic in situ hybridization (ISH) using InForm 1.4.0 software. We found diffuse nuclear expression of both HOTAIR and MALAT1 in all primary tumors of GEP-NENs with variable intensities. By multivariate model which adjusted for age and histologic grade, high expression of HOTAIR was associated with lower presenting T and M stages and subsequent development of metastases (P < 0.05). MALAT1 expression was associated with presenting T stage and development of metastases (P < 0.05). In summary, MALAT1 and HOTAIR are commonly expressed in GEP-NENs. High expression of either lncRNA showed grade-independent associations with clinically less aggressive disease.

A substantial proportion of patients infected with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) develop severe/critical coronavirus disease 2019 (COVID-19) characterized by acute respiratory distress syndrome (ARDS) with thrombosis.We tested the hypothesis that SARS-CoV-2--induced upregulation of tissue factor (TF) expression may be responsible for thrombus formation in COVID-19.We compared autopsy lung tissues from 11 patients with COVID-19--associated ARDS with samples from 6 patients with ARDS from other causes (non-COVID-19 ARDS) and 11 normal control lungs.Dual RNA in situ hybridization for SARS-CoV-2 and TF identified sporadic clustered SARS-CoV-2 with prominent co-localization of SARS-CoV-2 and TF RNA. TF expression was 2-fold higher in COVID-19 than in non-COVID-19 ARDS lungs (P = .017) and correlated with the intensity of SARS-CoV-2 staining (R2  = .36, P = .04). By immunofluorescence, TF protein expression was 2.1-fold higher in COVID-19 versus non-COVID-19 ARDS lungs (P = .0048) and 11-fold (P < .001) higher than control lungs. Fibrin thrombi and thrombi positive for platelet factor 4 (PF4) were found in close proximity to regions expressing TF in COVID-19 ARDS lung, and correlated with TF expression (fibrin, R2  = .52, P < .001; PF4, R2  = .59, P < .001).These data suggest that upregulation of TF expression is associated with thrombus formation in COVID-19 lungs and could be a key therapeutic target. Correlation of TF expression with SARS-CoV-2 in lungs of COVID-19 patients also raises the possibility of direct TF induction by the virus.

SARS-CoV-2 has been reported to show a capacity for invading the brains of humans and model animals. However, it remains unclear whether and how SARS-CoV-2 crosses the blood-brain barrier (BBB). Herein, SARS-CoV-2 RNA was occasionally detected in the vascular wall and perivascular space, as well as in brain microvascular endothelial cells (BMECs) in the infected K18-hACE2 transgenic mice. Moreover, the permeability of the infected vessel was increased. Furthermore, disintegrity of BBB was discovered in the infected hamsters by administration of Evans blue. Interestingly, the expression of claudin5, ZO-1, occludin and the ultrastructure of tight junctions (TJs) showed unchanged, whereas, the basement membrane was disrupted in the infected animals. Using an in vitro BBB model that comprises primary BMECs with astrocytes, SARS-CoV-2 was found to infect and cross through the BMECs. Consistent with in vivo experiments, the expression of MMP9 was increased and collagen IV was decreased while the markers for TJs were not altered in the SARS-CoV-2-infected BMECs. Besides, inflammatory responses including vasculitis, glial activation, and upregulated inflammatory factors occurred after SARS-CoV-2 infection. Overall, our results provide evidence supporting that SARS-CoV-2 can cross the BBB in a transcellular pathway accompanied with basement membrane disrupted without obvious alteration of TJs.

Cellular senescence of the retinal pigment epithelium (RPE) is thought to play an important role in vision-threatening retinal degenerative diseases, such as age-related macular degeneration (AMD). However, the single-cell RNA profiles of control RPE tissue and RPE tissue exhibiting cellular senescence are not well known. We have analyzed the single-cell transcriptomes of control mice and mice with low-dose doxorubicin (Dox)-induced RPE senescence (Dox-RPE). Our results have identified 4 main subpopulations in the control RPE that exhibit heterogeneous biological activities and play roles in ATP synthesis, cell mobility/differentiation, mRNA processing, and catalytic activity. In Dox-RPE mice, cellular senescence mainly occurs in the specific cluster, which has been characterized by catalytic activity in the control RPE. Furthermore, in the Dox-RPE mice, 6 genes that have not previously been associated with senescence also show altered expression in 4 clusters. Our results might serve as a useful reference for the study of control and senescent RPE.

Ventilatory support, such as supplemental oxygen, used to save premature infants impairs the growth of the pulmonary microvasculature and distal alveoli, leading to bronchopulmonary dysplasia (BPD). Although lung cellular composition changes with exposure to hyperoxia in neonatal mice, most human BPD survivors are weaned off oxygen within the first weeks to months of life, yet they may have persistent lung injury and pulmonary dysfunction as adults. We hypothesized that early-life hyperoxia alters the cellular landscape in later life and predicts long-term lung injury. Using single-cell RNA sequencing, we mapped lung cell subpopulations at postnatal day (pnd)7 and pnd60 in mice exposed to hyperoxia (95% O2) for 3 days as neonates. We interrogated over 10,000 cells and identified a total of 45 clusters within 32 cell states. Neonatal hyperoxia caused persistent compositional changes in later life (pnd60) in all five type II cell states with unique signatures and function. Premature infants requiring mechanical ventilation with different durations also showed similar alterations in these unique signatures of type II cell states. Pathologically, neonatal hyperoxic exposure caused alveolar simplification in adult mice. We conclude that neonatal hyperoxia alters the lung cellular landscape in later life, uncovering neonatal programing of adult lung dysfunction.

Cervical cancer is the leading cause of morbidity and mortality in women throughout the world, human papillomavirus 16 (HPV16) is the main type of HPV causing invasive cervical cancer. However, the underlying mechanism of the high carcinogenicity of HPV16 remains unclear. In the current study, we documented that metastasis-associated lung adenocarcinoma transcript 1 (MALAT1), a long noncoding RNA, is upregulated in HPV16-positive cervical cancer tissue and cell lines. The results of immunohistochemistry and immunofluorescence showed that MALAT1 was mainly localized in the cytoplasm. To clarify the biological functions of MALAT1 in cervical cancer cells, we performed gain- and loss-of-function experiments to explore the underlying molecular mechanism. Functionally, the proliferation of cervical cancer was detected by Cell Counting Kit-8 (CCK-8) and colony formation assay in MALAT1 overexpression or knockdown cells, our data showed that MALAT1 promotes the proliferation of cervical cancer cells. Mechanistically, our results suggested that MALAT1 upregulates Methionine adenosyltransferase 2A (MAT2A) by sponging miR-485-5p. Moreover, the gain-of-function assay validated the function of MAT2A in HPV16-positive cervical cancer proliferation. Taken together, our results demonstrated that MALAT1 acts as a competitive endogenous RNA (ceRNA) to regulate MAT2A by sponging miR-485-5p in HPV16-positive cervical cancer, suggesting that MALAT1 may act as a potential therapeutic target for HPV16-positive cervical cancer.

Chemosensory changes are well-reported symptoms of SARS-CoV-2 infection. The virus targets cells for entry by binding of its spike protein to cell-surface angiotensin-converting enzyme- 2 (ACE2). It was not known whether ACE2 is expressed on taste receptor cells (TRCs) nor if TRCs are infected directly. Using an in-situ hybridization (ISH) probe and an antibody specific to ACE2, ACE2 is present on a subpopulation of TRCs, namely, Type II cells in taste buds in taste papillae. Fungiform papillae (FP) of a SARS-CoV-2+ patient exhibiting symptoms of COVID-19, including taste changes, were biopsied. Based on ISH, replicating SARS-CoV-2 was present in Type II cells. Therefore, taste Type II cells provide a potential portal for viral entry that predicts vulnerabilities to SARS-CoV-2 in the oral cavity. The continuity and cell turnover of the patient's FP taste stem cell layer were disrupted during infection and had not completely recovered 6 weeks post symptom onset. Another patient suffering post-COVID-19 taste disturbances also had disrupted stem cells. These results demonstrate the possibility that novel and sudden taste changes frequently reported in COVID-19 may be the result of direct infection of taste papillae by SARS-CoV-2. This may result in impaired taste receptor stem cell activity and suggest more work is needed to understand the acute and post-acute dynamics of viral kinetics in the human taste bud.

BACKGROUND: With recorded under-performance of current standard therapeutic strategies as highlighted by high rates of post-treatment (resection or local ablation) recurrence, resistance to chemotherapy, poor overall survival, and an increasing global incidence, hepatocellular carcinoma (HCC) constitutes a medical challenge. Accumulating evidence implicates the presence of HCC stem cells (HCC-SCs) in HCC development, drug-resistance, recurrence, and progression. Therefore, treatment strategies targeting both HCC-SCs and non-CSCs are essential. METHODS: Recently, there has been an increasing suggestion of MALAT1 oncogenic activity in HCC; however, its role in HCC stemness remains unexplored. Herein, we investigated the probable role of MALAT1 in the SCs-like phenotype of HCC and explored likely molecular mechanisms by which MALAT1 modulates HCC-SCs-like and metastatic phenotypes. RESULTS: We showed that relative to normal, cirrhotic, or dysplastic liver conditions, MALAT1 was aberrantly expressed in HCC, similar to its overexpression in Huh7, Mahlavu, and SK-Hep1 HCC cells lines, compared to the normal liver cell line THLE-2. We also demonstrated a positive correlation between MALAT1 expression and poor cell differentiation status in HCC using RNAscope. Interestingly, we demonstrated that shRNA-mediated silencing of MALAT1 concomitantly downregulated the expression levels of ?-catenin, Stat3, c-Myc, CK19, vimentin, and Twist1 proteins, inhibited HCC oncogenicity, and significantly suppressed the HCC-SCs-related dye-effluxing potential of HCC cells and reduced their ALDH-1 activity, partially due to inhibited MALAT1-?-catenin interaction. Additionally, using TOP/FOP (TCL/LEF-Firefly luciferase) Flash, RT-PCR, and western blot assays, we showed that silencing MALAT1 downregulates ?-catenin expression, dysregulates the canonical Wnt signaling pathway, and consequently attenuates HCC tumorsphere formation efficiency, with concurrent reduction in CD133+ and CD90+ HCC cell population, and inhibits tumor growth in SK-Hep1-bearing mice. Conclusions: Taken together, our data indicate that MALAT1/Wnt is a targetable molecular candidate, and the therapeutic targeting of MALAT1/Wnt may constitute a novel promising anticancer strategy for HCC treatment.