Melanocortin receptor 4 as a new target in melanoma therapy: Anticancer activity of the inhibitor ML00253764 alone and in association with B-raf inhibitor vemurafenib.

The aim of our study is to investigate in vitro and in vivo MC4R as a novel target in melanoma using the selective antagonist ML00253764 (ML) alone and in combination with vemurafenib, a B-rafV600E inhibitor. The human melanoma B-raf mutated A-2058 and WM 266-4 cell lines were used. An MC4R null A-2058 cell line was generated using a CRISPR/Cas9 system. MC4R protein expression was analysed by western blotting, immunohistochemistry, and immunofluorescence. Proliferation and apoptotic assays were performed with ML00253764, whereas the synergism with vemurafenib was evaluated by the combination index (CI) and Loewe methods. ERK1/2 phosphorylation and BCL-XL expression were quantified by western blot. In vivo experiments were performed in Athymic Nude-Foxn1nu male mice, injecting subcutaneously melanoma cells, and treating animals with ML, vemurafenib and their concomitant combination. Comet and cytome assays were performed. Our results show that human melanoma cell lines A-2058 and WM 266-4, and melanoma human tissue, express functional MC4R receptors on their surface. MC4R receptors on melanoma cells can be inhibited by the selective antagonist ML, causing antiproliferative and proapoptotic activity through the inhibition of phosphorylation of ERK1/2 and a reduction of BCL-XL. The concomitant combination of vemurafenib and ML caused a synergistic effect on melanoma cells in vitro and inhibited in vivo tumor growth in a preclinical model, without causing mouse weight loss or genotoxicity. Our original research contributes to the landscape of pharmacological treatments for melanoma, providing MC4R antagonists as drugs that can be added to established therapies.

Effects of Nutraceuticals on Cisplatin-Induced Cytotoxicity in HEI-OC1 Cells.

Cisplatin is a chemotherapeutic drug for the treatment of several solid tumors, whose use is limited by its nephrotoxicity, neurotoxicity, ototoxicity, and development of resistance. The toxicity is caused by DNA cross-linking, increase in reactive oxygen species and/or depletion of cell antioxidant defenses. The aim of the work was to study the effect of antioxidant compounds (Lisosan G, Taurisolo®) or hydrogen sulfide (H2S)-releasing compounds (erucin) in the auditory HEI-OC1 cell line treated with cisplatin. Cell viability was determined using the MTT assay. Caspase and sphingomyelinase activities were measured by fluorometric and colorimetric methods, respectively. Expression of transcription factors, apoptosis hallmarks and genes codifying for antioxidant response proteins were measured by Western blot and/or RT-qPCR. Lisosan G, Taurisolo® and erucin did not show protective effects. Sodium hydrosulfide (NaHS), a donor of H2S, increased the viability of cisplatin-treated cells and the transcription of heme oxygenase 1, superoxide dismutase 2, NAD(P)H quinone dehydrogenase type 1 and the catalytic subunit of glutamate-cysteine ligase and decreased reactive oxygen species (ROS), the Bax/Bcl2 ratio, caspase-3, caspase-8 and acid sphingomyelinase activity. Therefore, NaHS might counteract the cytotoxic effect of cisplatin by increasing the antioxidant response and by reducing ROS levels and caspase and acid sphingomyelinase activity.

Anti-inflammatory reprogramming of microglia cells by metabolic modulators to counteract neurodegeneration; a new role for Ranolazine.

Microglia chronic activation is a hallmark of several neurodegenerative diseases, including the retinal ones, possibly contributing to their etiopathogenesis. However, some microglia sub-populations have anti-inflammatory and neuroprotective functions, thus making arduous deciphering the role of these cells in neurodegeneration. Since it has been proposed that functionally different microglia subsets also rely on different metabolic routes, we hypothesized that modulating microglia metabolism might be a tool to enhance their anti-inflammatory features. This would have a preventive and therapeutic potential in counteracting neurodegenerative diseases. For this purpose, we tested various molecules known to act on cell metabolism, and we revealed the anti-inflammatory effect of the FDA-approved piperazine derivative Ranolazine on microglia cells, while confirming the one of the flavonoids Quercetin and Naringenin, both in vitro and in vivo. We also demonstrated the synergistic anti-inflammatory effect of Quercetin and Idebenone, and the ability of Ranolazine, Quercetin and Naringenin to counteract the neurotoxic effect of LPS-activated microglia on 661W neuronal cells. Overall, these data suggest that using the selected molecules -also in combination therapies- might represent a valuable approach to reduce inflammation and neurodegeneration while avoiding long term side effects of corticosteroids.

Human T-Cell Leukemia Virus Type 1 Oncogenesis between Active Expression and Latency: A Possible Source for the Development of Therapeutic Targets.

The human T-cell leukemia virus type 1 (HTLV-1) is the only known human oncogenic retrovirus. HTLV-1 can cause a type of cancer called adult T-cell leukemia/lymphoma (ATL). The virus is transmitted through the body fluids of infected individuals, primarily breast milk, blood, and semen. At least 5-10 million people in the world are infected with HTLV-1. In addition to ATL, HTLV-1 infection can also cause HTLV-I-associated myelopathy (HAM/TSP). ATL is characterized by a low viral expression and poor prognosis. The oncogenic mechanism triggered by HTLV-1 is extremely complex and the molecular pathways are not fully understood. However, viral regulatory proteins Tax and HTLV-1 bZIP factor (HBZ) have been shown to play key roles in the transformation of HTLV-1-infected T cells. Moreover, several studies have shown that the final fate of HTLV-1-infected transformed Tcell clones is the result of a complex interplay of HTLV-1 oncogenic protein expression with cellular transcription factors that subvert the cell cycle and disrupt regulated cell death, thereby exerting their transforming effects. This review provides updated information on the mechanisms underlying the transforming action of HTLV-1 and highlights potential therapeutic targets to combat ATL.

Inhibiting immunoregulatory amidase NAAA blocks ZIKV maturation in Human Neural Stem Cells.

Recent evidence suggests that lipids play a crucial role in viral infections beyond their traditional functions of supplying envelope and energy, and creating protected niches for viral replication. In the case of Zika virus (ZIKV), it alters host lipids by enhancing lipogenesis and suppressing ß-oxidation to generate viral factories at the endoplasmic reticulum (ER) interface. This discovery prompted us to hypothesize that interference with lipogenesis could serve as a dual antiviral and anti-inflammatory strategy to combat the replication of positive sense single-stranded RNA (ssRNA+) viruses. To test this hypothesis, we examined the impact of inhibiting N-Acylethanolamine acid amidase (NAAA) on ZIKV-infected human Neural Stem Cells. NAAA is responsible for the hydrolysis of palmitoylethanolamide (PEA) in lysosomes and endolysosomes. Inhibition of NAAA results in PEA accumulation, which activates peroxisome proliferator-activated receptor-α (PPAR-α), directing ß-oxidation and preventing inflammation. Our findings indicate that inhibiting NAAA through gene-editing or drugs moderately reduces ZIKV replication by approximately one log10 in Human Neural Stem Cells, while also releasing immature virions that have lost their infectivity. This inhibition impairs furin-mediated prM cleavage, ultimately blocking ZIKV maturation. In summary, our study highlights NAAA as a host target for ZIKV infection.

Liver disease is a significant risk factor for cardiovascular outcomes - A UK Biobank study.

BACKGROUND & AIMS: Chronic liver disease (CLD) is associated with increased cardiovascular disease (CVD) risk. We investigated whether early signs of liver disease (measured by iron-corrected T1-mapping [cT1]) were associated with an increased risk of major CVD events. METHODS: Liver disease activity (cT1) and fat (proton density fat fraction [PDFF]) were measured using LiverMultiScan® between January 2016 and February 2020 in the UK Biobank imaging sub-study. Using multivariable Cox regression, we explored associations between liver cT1 (MRI) and primary CVD (coronary artery disease, atrial fibrillation [AF], embolism/vascular events, heart failure [HF] and stroke), and CVD hospitalisation and all-cause mortality. Liver blood biomarkers, general metabolism biomarkers, and demographics were also included. Subgroup analysis was conducted in those without metabolic syndrome (defined as at least three of: a large waist, high triglycerides, low high-density lipoprotein cholesterol, increased systolic blood pressure, or elevated haemoglobin A1c). RESULTS: A total of 33,616 participants (mean age 65 years, mean BMI 26 kg/m2, mean haemoglobin A1c 35 mmol/mol) had complete MRI liver data with linked clinical outcomes (median time to major CVD event onset: 1.4 years [range: 0.002-5.1]; follow-up: 2.5 years [range: 1.1-5.2]). Liver disease activity (cT1), but not liver fat (PDFF), was associated with higher risk of any major CVD event (hazard ratio 1.14; 95% CI 1.03-1.26; p = 0.008), AF (1.30; 1.12-1.51; p <0.001); HF (1.30; 1.09-1.56; p= 0.004); CVD hospitalisation (1.27; 1.18-1.37; p <0.001) and all-cause mortality (1.19; 1.02-1.38; p = 0.026). FIB-4 index was associated with HF (1.06; 1.01-1.10; p = 0.007). Risk of CVD hospitalisation was independently associated with cT1 in individuals without metabolic syndrome (1.26; 1.13-1.4; p <0.001). CONCLUSION: Liver disease activity, by cT1, was independently associated with a higher risk of incident CVD and all-cause mortality, independent of pre-existing metabolic syndrome, liver fibrosis or fat. IMPACT AND IMPLICATIONS: Chronic liver disease (CLD) is associated with a twofold greater incidence of cardiovascular disease. Our work shows that early liver disease on iron-corrected T1 mapping was associated with a higher risk of major cardiovascular disease (14%), cardiovascular disease hospitalisation (27%) and all-cause mortality (19%). These findings highlight the prognostic relevance of a comprehensive evaluation of liver health in populations at risk of CVD and/or CLD, even in the absence of clinical manifestations or metabolic syndrome, when there is an opportunity to modify/address risk factors and prevent disease progression. As such, they are relevant to patients, carers, clinicians, and policymakers.

Gene silencing of endothelial von Willebrand factor reduces the susceptibility of human endothelial cells to SARS-CoV-2 infection.

Mechanisms underlying vascular endothelial susceptibility to infection by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) are not fully understood. Emerging evidence indicates that patients lacking von Willebrand factor (vWF), an endothelial hallmark, are less severely affected by SARS-CoV-2 infection, yet the precise role of endothelial vWF in modulating coronavirus entry into endothelial cells is unknown. In the present study, we demonstrated that effective gene silencing by short interfering RNA (siRNA) for vWF expression in resting human umbilical vein endothelial cells (HUVECs) significantly reduced by 56% the cellular levels of SARS-CoV-2 genomic RNA. Similar reduction in intracellular SARS-CoV-2 genomic RNA levels was observed in non-activated HUVECs treated with siRNA targeting angiotensin-converting enzyme 2 (ACE2), the cellular gateway to coronavirus. By integrating quantitative information from real-time PCR and high-resolution confocal imaging, we demonstrated that ACE2 gene expression and its plasma membrane localization in HUVECs were both markedly reduced after treatment with siRNA anti-vWF or anti-ACE2. Conversely, siRNA anti-ACE2 did not reduce endothelial vWF gene expression and protein levels. Finally, SARS-CoV-2 infection of viable HUVECs was enhanced by overexpression of vWF, which increased ACE2 levels. Of note, we found a similar increase in interferon-ß mRNA levels following transfection with untargeted, anti-vWF or anti-ACE2 siRNA and pcDNA3.1-WT-VWF. We envision that siRNA targeting endothelial vWF will protect against productive endothelial infection by SARS-CoV-2 through downregulation of ACE2 expression and might serve as a novel tool to induce disease resistance by modulating the regulatory role of vWF on ACE2 expression.

Canonical fibroblast growth factors in viral infection.

Fibroblast growth factors (FGFs) are a family of proteins that play a crucial role in the development and maintenance of various tissues in the body. There are three function-al groups of FGFs: canonical FGFs (cFGFs), intracellularly retained FGFs, and metabolic (also called endocrine) FGFs. cFGFs are secreted and act in an autocrine/paracrine fashion to regulate differentiation during foetal development, as well as tissue repair in adults. Recent studies have also begun to unravel the role of cFGFs during viral infections, suggesting that FGF-2 and other canonical FGFs may have an important virus-specific role, also by the regulation of the immune response. Because dysregulation in the FGF pathways is pivotal in cancer development, FGFs are the target of many anticancer drugs. These drugs may be repurposed to treat viral infection, since dysregulation of FGF signalling has been implicated in the pathogenesis of viral infections, such as hepatitis C. Overall, the role of cFGFs during viral infection is an underrepresented area of current research. This review focuses on overviewing the effects of canonical FGFs during infection by different viruses. Many studies highlight that the effects of FGFs during viral infection may be complex and context-dependent. While there is evidence to suggest that FGFs may have a beneficial impact on the immune response and tissue repair during viral infection, further studies are needed to fully understand the mechanisms underlying these effects and to determine in what cases FGFs could be targeted as a therapeutic approach for viral infection.

Targeting SIRT1 Rescues Age- and Obesity-Induced Microvascular Dysfunction in Ex Vivo Human Vessels.

BACKGROUND: Experimental evidence suggests a key role of SIRT1 (silent information regulator 1) in age- and metabolic-related vascular dysfunction. Whether these effects hold true in the human microvasculature is unknown. We aimed to investigate the SIRT1 role in very early stages of age- and obesity-related microvascular dysfunction in humans. METHODS: Ninety-five subjects undergoing elective laparoscopic surgery were recruited and stratified based on their body mass index status (above or below 30 kg/m2) and age (above or below 40 years) in 4 groups: Young Nonobese, Young Obese, Old Nonobese, and Old Obese. We measured small resistance arteries' endothelial function by pressurized micromyography before and after incubation with a SIRT1 agonist (SRT1720) and a mitochondria reactive oxygen species (mtROS) scavenger (MitoTEMPO). We assessed vascular levels of mtROS and nitric oxide availability by confocal microscopy and vascular gene expression of SIRT1 and mitochondrial proteins by qPCR. Chromatin immunoprecipitation assay was employed to investigate SIRT1-dependent epigenetic regulation of mitochondrial proteins. RESULTS: Compared with Young Nonobese, obese and older patients showed lower vascular expression of SIRT1 and antioxidant proteins (FOXO3 [forkhead box protein O3] and SOD2) and higher expression of pro-oxidant and aging mitochondria proteins p66Shc and Arginase II. Old Obese, Young Obese and Old Nonobese groups endothelial dysfunction was rescued by SRT1720. The restoration was comparable to the one obtained with mitoTEMPO. These effects were explained by SIRT1-dependent chromatin changes leading to reduced p66Shc expression and upregulation of proteins involved in mitochondria respiratory chain. CONCLUSIONS: SIRT1 is a novel central modulator of the earliest microvascular damage induced by age and obesity. Through a complex epigenetic control mainly involving p66Shc and Arginase II, it influences mtROS levels, NO availability, and the expression of proteins of the mitochondria respiratory chain. Therapeutic modulation of SIRT1 restores obesity- and age-related endothelial dysfunction. Early targeting of SIRT1 might represent a crucial strategy to prevent age- and obesity-related microvascular dysfunction.

Design, synthesis, and biological screening of a series of 4'-fluoro-benzotriazole-acrylonitrile derivatives as microtubule-destabilising agents (MDAs).

Introduction: Colchicine-binding site inhibitors are some of the most interesting ligands belonging to the wider family of microtubule-destabilising agents.Results: A novel series of 4'-fluoro-substituted ligands (5-13) was synthesised. The antiproliferative activity assays resulted in nM values for the new benzotriazole-acrylonitrile derivatives. Compound 5, the hit compound, showed an evident blockade of HeLa cell cycle in the G2-M phase, but also a pro-apoptotic potential, and an increase of early and late apoptotic cells in HeLa and MCF-7 cell cycle analysis. Confocal microscopy analysis showed a segmented shape and a collapse of the cytoskeleton, as well as a consistent cell shrinkage after administration of 5 at 100 nM. Derivative 5 was also proved to compete with colchicine at colchicine-binding site, lowering its activity against tubulin polymerisation. In addition, co-administration of 5 and doxorubicin in drug-resistant A375 melanoma cell line highlighted a synergic potential in terms of inhibition of cell viability.Discussion: The 4'-fluoro substitution of benzotriazole-acrylonitrile scaffold brought us a step forward in the optimisation process to obtain compound 5 as promising MDA antiproliferative agent at nanomolar concentration.

Lipid balance remodelling by human positive-strand RNA viruses and the contribution of lysosomes.

A marked reorganization of internal membranes occurs in the cytoplasm of cells infected by single stranded positive-sense RNA viruses. Most cell compartments change their asset to provide lipids for membrane rearrangement into replication organelles, where to concentrate viral proteins and enzymes while hiding from pathogen pattern recognition molecules. Because the endoplasmic reticulum is a central hub for lipid metabolism, when viruses hijack the organelle to form their replication organelles, a cascade of events change the intracellular environment. This results in a marked increase in lipid consumption, both by lipolysis and lipophagy of lipid droplets. In addition, lipids are used to produce energy for viral replication. At the same time, inflammation is started by signalling lipids, where lysosomal processing plays a relevant role. This review is aimed at providing an overview on what takes place after human class IV viruses have released their genome into the host cell and the consequences on lipid metabolism, including lysosomes.

Zika virus induces FOXG1 nuclear displacement and downregulation in human neural progenitors.

Congenital alterations in the levels of the transcription factor Forkhead box g1 (FOXG1) coding gene trigger "FOXG1 syndrome," a spectrum that recapitulates birth defects found in the "congenital Zika syndrome," such as microcephaly and other neurodevelopmental conditions. Here, we report that Zika virus (ZIKV) infection alters FOXG1 nuclear localization and causes its downregulation, thus impairing expression of genes involved in cell replication and apoptosis in several cell models, including human neural progenitor cells. Growth factors, such as EGF and FGF2, and Thr271 residue located in FOXG1 AKT domain, take part in the nuclear displacement and apoptosis protection, respectively. Finally, by progressive deletion of FOXG1 sequence, we identify the C-terminus and the residues 428-481 as critical domains. Collectively, our data suggest a causal mechanism by which ZIKV affects FOXG1, its target genes, cell cycle progression, and survival of human neural progenitors, thus contributing to microcephaly.

Palmitoylethanolamide (PEA) Inhibits SARS-CoV-2 Entry by Interacting with S Protein and ACE-2 Receptor.

Lipids play a crucial role in the entry and egress of viruses, regardless of whether they are naked or enveloped. Recent evidence shows that lipid involvement in viral infection goes much further. During replication, many viruses rearrange internal lipid membranes to create niches where they replicate and assemble. Because of the close connection between lipids and inflammation, the derangement of lipid metabolism also results in the production of inflammatory stimuli. Due to its pivotal function in the viral life cycle, lipid metabolism has become an area of intense research to understand how viruses seize lipids and to design antiviral drugs targeting lipid pathways. Palmitoylethanolamide (PEA) is a lipid-derived peroxisome proliferator-activated receptor-α (PPAR-α) agonist that also counteracts SARS-CoV-2 entry and its replication. Our work highlights for the first time the antiviral potency of PEA against SARS-CoV-2, exerting its activity by two different mechanisms. First, its binding to the SARS-CoV-2 S protein causes a drop in viral infection of ~70%. We show that this activity is specific for SARS-CoV-2, as it does not prevent infection by VSV or HSV-2, other enveloped viruses that use different glycoproteins and entry receptors to mediate their entry. Second, we show that in infected Huh-7 cells, treatment with PEA dismantles lipid droplets, preventing the usage of these vesicular bodies by SARS-CoV-2 as a source of energy and protection against innate cellular defenses. This is not surprising since PEA activates PPAR-α, a transcription factor that, once activated, generates a cascade of events that leads to the disruption of fatty acid droplets, thereby bringing about lipid droplet degradation through ß-oxidation. In conclusion, the present work demonstrates a novel mechanism of action for PEA as a direct and indirect antiviral agent against SARS-CoV-2. This evidence reinforces the notion that treatment with this compound might significantly impact the course of COVID-19. Indeed, considering that the protective effects of PEA in COVID-19 are the current objectives of two clinical trials (NCT04619706 and NCT04568876) and given the relative lack of toxicity of PEA in humans, further preclinical and clinical tests will be needed to fully consider PEA as a promising adjuvant therapy in the current COVID-19 pandemic or against emerging RNA viruses that share the same route of replication as coronaviruses.

Patterns of neutralizing humoral response to SARS-CoV-2 infection among hematologic malignancy patients reveal a robust immune response in anti-cancer therapy-naive patients.

Understanding antibody-based SARS-CoV-2 immunity in hematologic malignancy (HM) patients following infection is crucial to inform vaccination strategies for this highly vulnerable population. This cross-sectional study documents the anti-SARS-CoV-2 humoral response and serum neutralizing activity in 189 HM patients recovering from a PCR-confirmed infection. The overall seroconversion rate was 85.7%, with the lowest values in patients with lymphoid malignancies or undergoing chemotherapy. Therapy-naive patients in the "watch and wait" status were more likely to seroconvert and display increased anti-s IgG titers. Enhanced serum neutralizing activity was observed in the following SARS-CoV-2-infected HM patient groups: (i) males; (ii) severe COVID-19; and (iii) "watch and wait" or "complete/partial response". The geometric mean (GeoMean) ID50 neutralization titers in patients analyzed before or after 6 months post-infection were 299.1 and 306.3, respectively, indicating that >50% of the patients in either group had a neutralization titer sufficient to provide 50% protection from symptomatic COVID-19. Altogether, our findings suggest that therapy-naive HM patients mount a far more robust immune response to SARS-CoV-2 infection vs. patients receiving anti-cancer treatment, raising the important question as to whether HM patients should be vaccinated before therapy and/or receive vaccine formats capable of better recapitulating the natural infection.

A low-cost simple test for weekly detection of Mycoplasma hyorhinis and arginini contaminations in cell cultures and viral preparations.

Mollicutes (Mycoplasma and Acholeplasma) are parasitic bacteria that adhere to cellular surfaces, naturally resistant to many antibiotics and extremely small. They are often found as contaminants in cultured cells, where they go unnoticed. They may be present in viral stocks because they are present in supernatants of cells where cultured viruses are released. The best way to keep laboratories free of Mycoplasma is to discard infected cultures, but, as judged by the very common finding of Mycoplasma-contaminated cultures in many laboratories, this is not done as often as it should be. A possible reason is that most procedures recommended take as long as performing a simple experiment and many laboratories delay testing to save money and time. Indeed, many methods exist to detect Mycoplasma infection of cell lines, but they take at least a couple of hours of hands-on work, if not more. Here we describe a procedure to screen viral stocks and tissue cultures for Mycoplasma presence. It relies on isolation of Mycoplasma on ordinary horse blood agar directly from exhausted tissue culture supernatants and does not require experienced personnel or expensive equipment. It only requires minutes of hands-on work, and, for this, it may be useful for weekly screening of cultures. It yields semiquantitative results in roughly 5 days, which is the time that usually passes between one subculture passage of cells in vitro to another. Because of its simplicity, it may be useful for detecting Mycoplasma in viral stocks and for frequent screening of cultures in research laboratories.

Evolution of viruses and the emergence of SARS-CoV-2 variants.

Life implies adaptation. This is one of the fundamental principles that has permitted most living species to survive through ages in an ever-changing environment. Spontaneously occurring events have shaped also virus populations and their fitness. Thanks to their plasticity, viruses have thrived in extremely dissimilar conditions. Unsurprisingly, SARS-CoV-2, the etiological agent of COVID-19, is no exception. Thanks to an unprecedented rate of molecular tracing and sequence scrutiny, the virus was followed in all its changes and shown to evolve in such a way as to possibly determine subsequent waves of infection after the first global and massive outbreak. This review illustrates the major modifications occurred to the virus since its discovery. We describe the potential advantages that these changes conveyed as regards SARS-CoV-2 transmissibility, resistance to host innate and adaptive barriers and molecular diagnosis.

PCSK9 Induces Tissue Factor Expression by Activation of TLR4/NFkB Signaling.

Proprotein convertase subtilisin kexin 9 (PCSK9) increases LDL cholesterol (C) concentration by accelerating the hepatic degradation of the LDL receptor (R) thus promoting atherogenesis. The molecule, however, also exerts proinflammatory effects independent of circulating LDL-C by enhancing local cytokine production and activation of NFkB, a process that might involve Toll-like receptor 4 (TLR4), a crucial component of the innate immunity system. Tissue factor (TF), a glycoprotein which plays an essential role in coagulation and inflammation, is rapidly induced by circulating monocytes stimulated by proinflammatory agents through NFkB-dependent mechanisms. The aims of our study were (1) to assess whether PCSK9 may induce monocytic TF expression and (2) to evaluate whether the TLR4/NFkB signaling pathway may contribute to that effect. Experiments were carried out in peripheral blood mononuclear cells (PBMCs), THP-1 cells, and HEK293 cells transfected with plasmids encoding the human TLR4 complex. PCSK9 increased procoagulant activity (PCA), mRNA and TF protein expression in both PBMCs and THP-1 cultures. Pre-treatment with inhibitors of TLR4/NFkB signaling such as LPS-RS, CLI-095, and BAY 11-7082, downregulated PCSK9-induced TF expression. A similar effect was obtained by incubating cell cultures with anti-PCSK9 human monoclonal antibody. In TLR4-HEK293 cells, PCSK9 activated the TLR4/NFkB signaling pathway to an extent comparable to LPS, the specific agonist of TLR4s and quantitative confocal microscopy documented the colocalization of PCSK9 and TLR4s. In conclusion, PCSK9 induces TF expression through activation of TLR4/NFkB signaling.

A spatial multi-scale fluorescence microscopy toolbox discloses entry checkpoints of SARS-CoV-2 variants in Vero E6 cells.

We exploited a multi-scale microscopy imaging toolbox to address some major issues related to SARS-CoV-2 interactions with host cells. Our approach harnesses both conventional and super-resolution fluorescence microscopy and easily matches the spatial scale of single-virus/cell checkpoints. After its validation through the characterization of infected cells and virus morphology, we leveraged this toolbox to reveal subtle issues related to the entry phase of SARS-CoV-2 variants in Vero E6 cells. Our results show that in Vero E6 cells the B.1.1.7 strain (aka Alpha Variant of Concern) is associated with much faster kinetics of endocytic uptake compared to its ancestor B.1.177. Given the cell-entry scenario dominated by the endosomal "late pathway", the faster internalization of B.1.1.7 could be directly related to the N501Y mutation in the S protein, which is known to strengthen the binding of Spike receptor binding domain with ACE2. Remarkably, we also directly observed the central role of clathrin as a mediator of endocytosis in the late pathway of entry. In keeping with the clathrin-mediated endocytosis, we highlighted the non-raft membrane localization of ACE2. Overall, we believe that our fluorescence microscopy-based approach represents a fertile strategy to investigate the molecular features of SARS-CoV-2 interactions with cells.