Antithrombin, the main physiological inhibitor of the coagulation cascade, exerts anti-tumor effects on glioblastoma multiforme cells. Antithrombin has different conformations: native, heparin-activated, prelatent, latent, and cleaved. The prelatent form has an intermediate affinity between latent and native antithrombin, although it is the most antiangiogenic form. Herein, we investigate the effect of this conformation on the tumorigenic processes of glioblastoma multiforme cells. Antithrombin forms were purified by chromatography. Chromogenic/fluorogenic assays were carried out to evaluate enteropeptidase and hepsin inhibition, two serine proteases involved in these processes. Wound healing, Matrigel invasion and BrdU incorporation assays were performed to study migration, invasion and proliferation. E-cadherin, Vimentin, VEGFA, pAKT, STAT3, pSTAT3, and pERK1/2 expression was assessed by Western blot and/or qRT-PCR. Prelatent antithrombin inhibited both enteropeptidase and hepsin, although it was less efficient than the native conformation. Exposure to prelatent antithrombin significantly reduced migration and invasion but not proliferation of U-87 MG, being the conformation most efficient on migration. Prelatent antithrombin down-regulated VEGFA, pSTAT3, and pERK1/2 expression in U-87 MG cells. Our work elucidates that prelatent antithrombin has surprisingly versatile anti-tumor properties in U-87 MG glioblastoma multiforme cells. This associates with resistance pathway activation, the decreased expression of tumorigenic proteins, and increased angiogenesis, postulating the existence of a new, formerly unknown receptor with potential therapeutic implications. Full article

This study aims to evaluate whether mitochondrial changes occur in the early stages of bipolar disorder (BD). Using fibroblasts derived from BD patients and matched controls, the levels of proteins involved in mitochondrial biogenesis and dynamics (fission and fusion) were evaluated by Western Blot analysis. Mitochondrial membrane potential (MMP) was studied using the fluorescent probe TMRE. Mitochondrial morphology was analyzed with the probe Mitotracker Green and mitophagy was evaluated by quantifying the co-localization of HSP60 (mitochondria marker) and LC3B (autophagosome marker) by immunofluorescence. Furthermore, the activity of the mitochondrial respiratory chain and the glycolytic capacity of controls and BD patients-derived cells were also studied using the Seahorse technology. BD patient-derived fibroblasts exhibit fragmented mitochondria concomitantly with changes in mitochondrial dynamics and biogenesis in comparison with controls. Moreover, a decrease in the MMP and increased mitophagy was observed in fibroblasts obtained from BD patients when compared with control cells. Impaired energetic metabolism due to inhibition of the mitochondrial electron transport chain (ETC) and subsequent ATP depletion, associated with glycolysis stimulation, was also a feature of BD fibroblasts. Overall, these results support the fact that mitochondrial disturbance is an early event implicated in BD pathophysiology that might trigger neuronal changes and modification of brain circuitry. Full article

Carotid artery plaques are considered a measure of atherosclerosis and are associated with an increased risk of atherosclerotic cardiovascular disease, particularly ischemic strokes. Monitoring of patients with an elevated risk of stroke is critical in developing better prevention strategies. Non-invasive imaging allows us to directly see atherosclerosis in vessels and many features that are related to plaque vulnerability. A large body of evidence has demonstrated a strong correlation between some lipid parameters and carotid atherosclerosis. In this article, we review the relationship between lipids and atherosclerosis with a focus on carotid ultrasound, the most common method to estimate atherosclerotic load. Full article

The current health crisis caused by coronavirus 2019 (COVID-19) and associated pathogens emphasize the urgent need for vaccine systems that can generate protective and long-lasting immune responses. Vaccination, employing peptides, nucleic acids, and other molecules, or using pathogen-based strategies, in fact, is one of the most potent approaches in the management of viral diseases. However, the vaccine candidate requires protection from degradation and precise delivery to the target cells. This can be achieved by employing different types of drug and vaccine delivery strategies, among which, nanotechnology-based systems seem to be more promising. This entry aims to provide insight into major aspects of vaccine design and formulation to address different diseases, including the recent outbreak of SARS-CoV-2. Special emphasis of this review is on the technical and practical aspects of vaccine construction and theranostic approaches to precisely target and localize the active compounds. Full article

Immunonutrition appears as a field with great potential in modern medicine. Since the immune system can trigger serious pathophysiological disorders, it is essential to study and implement a type of nutrition aimed at improving immune system functioning and reinforcing it individually for each patient. In this sense, the nucleotide-binding oligomerization domain-1 (NOD1), one of the members of the pattern recognition receptors (PRRs) family of innate immunity, has been related to numerous pathologies, such as cancer, diabetes, or cardiovascular diseases. NOD1, which is activated by bacterial-derived peptidoglycans, is known to be present in immune cells and to contribute to inflammation and other important pathways, such as fibrosis, upon recognition of its ligands. Since immunonutrition is a significant developing research area with much to discover, we propose NOD1 as a possible target to consider in this field. It is relevant to understand the cellular and molecular mechanisms that modulate the immune system and involve the activation of NOD1 in the context of immunonutrition and associated pathological conditions. Surgical or pharmacological treatments could clearly benefit from the synergy with specific and personalized nutrition that even considers the health status of each subject. Full article

The prevalence of metabolic disorders characterized by chronic inflammation has been on a sharp rise for decades. As such, tools that address metabolic and inflammatory dysregulation are of great importance. Plant stanols are well-known for reducing intestinal cholesterol absorption and may also have direct anti-inflammatory effects. In this study, our aim was to investigate to what extent the benefits of dietary plant stanol supplementation depend on dietary cholesterol intake in an experimental mouse model for cholesterol-induced metabolic inflammation. Here, we used Ldlr^−/− mice transplanted with Npc1^nih-derived bone marrow, featuring feature bone marrow-derived immune cells characterized by chronic inflammation induced by lysosomal lipid accumulation. Npc1^nih- and Npc1^wt-transplanted mice were placed on either a high fat, high cholesterol (HFC) or on a chow diet low in cholesterol, with or without 2% plant stanols supplementation. At the end of the study, the metabolic and inflammatory status of the mice was analyzed. Plant stanol supplementation to the HFC diet reduced liver cholesterol levels and improved lipid metabolism and liver inflammation, particularly in Npc1^nih-tp mice. In contrast, plant stanol supplementation to the chow diet did not significantly improve the aforementioned parameters, though similar reductive trends to those in the HFC diet setting were observed regarding liver cholesterol accumulation and liver inflammatory markers. The effects of dietary plant stanol supplementation on dietary cholesterol-induced inflammation are largely dependent on dietary cholesterol intake. Future research should verify whether other models of metabolic inflammation exhibit similar stanol-related effects on inflammation. Full article

“Where there is light, there must be shadow, [...] Full article

Bacillus Calmette-Guerin (BCG) and the cell wall skeleton (CWS) derived from BCG are known to enhance nonspecific immune activation and anti-cancer immunity; however, their roles as a vaccine adjuvant are largely unknown. Here, we report that BCG-CWS acts as a strong immune adjuvant by promoting the protective immune responses in mouse models with influenza vaccination. The different aged mice immunized with inactivated split vaccine with or without BCG-CWS were challenged with an influenza pandemic virus. When protective immune responses were compared, even a single immunization of adult mice with a BCG-CWS-adjuvanted vaccine showed significantly enhanced humoral immune responses with increased IgG1 and IgG2a isotype antibodies. Importantly, the protective effects by the BCG-CWS adjuvant for influenza vaccination upon humoral and cellular immunogenicity were comparable between infants (6 days and 2 weeks old) and aged (20 months old) mice. Moreover, BCG-CWS dramatically augmented vaccine-mediated protective responses, including decreased viral loads, lung damage, and airway resistance, as well as increased mouse survival, amelioration of weight loss, and proinflammatory cytokine expression in all experimental groups including infant, adults, and old aged mice. We further provided the evidence that the BCG-CWS adjuvant effects were mediated through Toll-like receptors (TLR) 2 and TLR4 signaling pathways. Together, these data suggest that BCG-CWS can be promising as a potential influenza vaccine adjuvant in both young and old aged population through TLR2/4-mediated immune-boosting activities. Full article

HOX genes appear to play a role in breast cancer progression in a molecular subtype-dependent way. The altered expression of HOXB7, for example, was reported to promote breast cancer progression in specific subtypes. Here we induced HOXB7 overexpression in MDA-MB-231 cells, a cellular model of the Triple-Negative breast cancer molecular subtype, and evaluated the phenotypic changes in cell viability, morphogenesis, migration, invasion, and colony formation. During the phenotypic characterization of the HOXB7-overexpressing cells, we consistently found less aggressive behavior represented by lower cell viability, inhibition of cell migration, invasion, and attachment-independent colony formation capacities added to the more compact and organized spheroid growth in 3D cultures. We then evaluated the expression of putative downstream targets and their direct binding to HOXB7 comparing ChIP-qPCR data generated from HOXB7-overexpressing cells and controls. In the manipulated cells, we found enriched biding of HOXB7 to CTNNB1, EGFR, FGF2, CDH1, DNMT3B, TGFB2, and COMMD7. Taken together, these results highlight the plasticity of the HOXB7 function in breast cancer, according to the cellular genetic background and expression levels, and provide evidence that in Triple-Negative breast cancer cells, HOXB7 overexpression has the potential to promote less aggressive phenotypes. Full article

Mutations in the PARK2 gene encoding the protein parkin cause autosomal recessive juvenile parkinsonism (ARJP), a neurodegenerative disease characterized by early dysfunction and loss of dopamine (DA) neurons in the substantia nigra pars compacta (SNc). No therapy is currently available to prevent or slow down the neurodegeneration in ARJP patients. Preclinical models are key to clarifying the early events that lead to neurodegeneration and reveal the potential of novel neuroprotective strategies. ParkinQ311X is a transgenic mouse model expressing in DA neurons a mutant parkin variant found in ARJP patients. This model was previously reported to show the neuropathological hallmark of the disease, i.e., the progressive loss of DA neurons. However, the early dysfunctions that precede neurodegeneration have never been investigated. Here, we analyzed SNc DA neurons in parkinQ311X mice and found early features of mitochondrial dysfunction, extensive cytoplasmic vacuolization, and dysregulation of spontaneous in vivo firing activity. These data suggest that the parkinQ311X mouse recapitulates key features of ARJP and provides a useful tool for studying the neurodegenerative mechanisms underlying the human disease and for screening potential neuroprotective drugs. Full article

Cardiovascular disease (CVD) and cancer are two major causes of death worldwide. The question is, “Could there be a link between these two pathologies in addition to their shared, common risk factors?” To find some answers, we studied the effect of oxidized low-density lipoproteins (oxLDL) on head and neck cancer (HNC) cell lines, since oxLDL is a major contributor to atherosclerosis and the principal cause of CVD. In this study, we exposed three HNC cell lines (Detroit 562, UPCI-SCC-131 and FaDu) to oxLDL. We investigated two oxLDL receptors, CD36 and Lox-1, using immunofluorescence. Cancer cell migration was evaluated using Boyden chambers and the Wnt/β-catenin pathway was investigated using Western blotting. We demonstrated that the expression of CD36 and Lox-1 significantly increases after exposure to oxLDL. Moreover, we found that oxLDL reduces the migration of HNC cell lines, an observation that is in line with an increased degradation of β-catenin under oxLDL. Finally, the inhibition of CD36 with sulfosuccinimidyl oleate (SSO) reverses the inhibition of cell migration. In conclusion, we report that oxLDL seems to induce an increase in CD36 expression on HNC cell lines, enhancing the uptake of these lipids in cells to finally decrease cancer cell migration via the CD36/β-catenin pathway. Full article

Antisense oligonucleotides (ASOs), siRNA and splice switching oligonucleotides (SSOs) all have immense potential as therapeutic agents, potential that is now being validated as oligonucleotides enter the clinic. However, progress in oligonucleotide-based therapeutics has been limited by the difficulty in delivering these complex molecules to their sites of action in the cytosol or nucleus of cells within specific tissues. There are two aspects to the delivery problem. The first is that most types of oligonucleotides have poor uptake into non-hepatic tissues. The second is that much of the oligonucleotide that is taken up by cells is entrapped in endosomes where it is pharmacologically inert. It has become increasingly recognized that endosomal trapping is a key constraint on oligonucleotide therapeutics. Thus, many approaches have been devised to address this problem, primarily ones based on various nanoparticle technologies. However, recently an alternative approach has emerged that employs small molecules to manipulate intracellular trafficking processes so as to enhance oligonucleotide actions. This review presents the current status of this chemical biology approach to oligonucleotide delivery and seeks to point out possible paths for future development. Full article

Messenger RNA is rapidly gaining significance as a therapeutic modality. Here, we address the dependence of dose–response functions on the type of delivery vehicle, cell line, and incubation time. Knowledge of these characteristics is crucial for the application of mRNA. As delivery vehicles, a lipid-based formulation and the cell-penetrating peptide Pepfect14 (PF14) were employed. As cell lines, we included a glomerular endothelial cell line (mGEnC) as a model for differentiated cells, HeLa cells, and SKOV-3 ovarian carcinoma cells. Uptake and expression were detected by flow cytometry, using a Cy5-labelled mRNA coding for enhanced green fluorescent protein (EGFP). There was a linear correlation of dose, uptake, and expression, and this correlation was maintained for over up to 72 h. Through application of a multistep kinetic model, we show that differences in expression levels can already be explained by the number of mRNAs packaged per delivery vehicle. Using luciferase as a reporter protein, linearity of expression was observed over 5 orders of magnitude in vitro and 3 orders of magnitude in vivo. Overall, the results demonstrate that mRNA provides excellent quantitative control over protein expression, also over extended periods of time. Full article

During a search for glycosidase inhibitors among marine natural products, we applied an integrated in vitro and in silico approach to evaluate the potency of some aaptamines and makaluvamines isolated from marine sponges on the hydrolyzing activity of α-N-acetylgalactosaminidase (α-NaGalase) from human cancer cells and the recombinant α-D-galactosidase (α-PsGal) from a marine bacterium Pseudoalteromonas sp. KMM 701. These alkaloids showed no direct inhibitory effect on the cancer α-NaGalase; but isoaaptamine (2), 9-demethylaaptamine (3), damirone B (6), and makaluvamine H (7) reduced the expression of the enzyme in the human colorectal adenocarcinoma cell line DLD-1 at 5 μM. Isoaaptamine (2), 9-demethylaaptamine (3), makaluvamine G (6), and zyzzyanone A (7) are slow-binding irreversible inhibitors of the bacterial α-PsGal with the inactivation rate constants (k_inact) 0.12 min⁻¹, 0.092 min⁻¹, 0.079 min⁻¹, and 0.037 min⁻¹, as well as equilibrium inhibition constants (K_i) 2.70 µM, 300 µM, 411 µM, and 105 µM, respectively. Docking analysis revealed that these alkaloids bind in a pocket close to the catalytic amino acid residues Asp451 and Asp516 and form complexes, due to π-π interactions with the Trp308 residue and hydrogen bonds with the Lys449 residue. None of the studied alkaloids formed complexes with the active site of the human α-NaGalase. Full article

Treatment with calcitriol, the hormonal form of vitamin D, has shown beneficial effects in experimental models of acute lung injury. In this study, we aimed to analyze the associations between calcitriol supplementation and the risk of SARS-CoV2 infection or COVID-19 mortality. Individuals ≥18 years old living in Catalonia and supplemented with calcitriol from April 2019 to February 2020 were compared with propensity score matched controls. Outcome variables were SARS-CoV2 infection, severe COVID-19 and COVID-19 mortality. Associations between calcitriol supplementation and outcome variables were analyzed using multivariable Cox proportional regression. A total of 8076 patients were identified as being on calcitriol treatment. Advanced chronic kidney disease and hypoparathyroidism were the most frequent reasons for calcitriol supplementation in our population. Calcitriol use was associated with reduced risk of SARS-CoV2 infection (HR 0.78 [CI 95% 0.64–0.94], p = 0.010), reduced risk of severe COVID-19 and reduced COVID-19 mortality (HR 0.57 (CI 95% 0.41–0.80), p = 0.001) in patients with advanced chronic kidney disease. In addition, an inverse association between mean daily calcitriol dose and COVID-19 severity or mortality was observed in treated patients, independently of renal function. Our findings point out that patients with advanced chronic kidney disease could benefit from calcitriol supplementation during the COVID-19 pandemic. Full article