International Journal of Molecular Sciences

Melanoma is the most aggressive and dangerous form of skin cancer that develops from transformed melanocytes. It is crucial to identify melanoma at its early stages, in situ, as it is “curable” at this stage. However, after metastasis, it is difficult to treat and the five-year survival is only 25%. In recent years, a better understanding of the etiology of melanoma and its progression has made it possible for the development of targeted therapeutics, such as vemurafenib and immunotherapies, to treat advanced melanomas. In this review, we focus on the molecular mechanisms that mediate melanoma development and progression, with a special focus on the immune evasion strategies utilized by melanomas, to evade host immune surveillances. The proposed mechanism of action and the roles of immunotherapeutic agents, ipilimumab, nivolumab, pembrolizumab, and atezolizumab, adoptive T- cell therapy plus T-VEC in the treatment of advanced melanoma are discussed. In this review, we implore that a better understanding of the steps that mediate melanoma onset and progression, immune evasion strategies exploited by these tumor cells, and the identification of biomarkers to predict treatment response are critical in the design of improved strategies to improve clinical outcomes for patients with this deadly disease. Full article

Glioblastoma is a type of aggressive brain tumor that grows very fast and evades surrounding normal brain, lead to treatment failure. Glioblastomas are associated with Akt activation due to somatic alterations in PI3 kinase/Akt pathway and/or PTEN tumor suppressor. Sodium meta-arsenite, KML001 is an orally bioavailable, water-soluble, and trivalent arsenical and it shows antitumoral effects in several solid tumor cells via inhibiting oncogenic signaling, including Akt and MAPK. Here, we evaluated the effect of sodium meta-arsenite, KML001, on the growth of human glioblastoma cell lines with different PTEN expression status and Akt activation, including PTEN-deficient cells (U87-MG and U251) and PTEN-positive cells (LN229). The growth-inhibitory effect of KML001 was stronger in U87-MG and U251 cells, which exhibited higher Akt activity than LN229 cells. KML001 deactivated Akt and decreased its protein levels via proteasomal degradation in U87-MG cells. KML001 upregulated mutant PTEN levels via inhibition of its proteasomal degradation. KML001 inhibited cell growth more effectively in active Akt-overexpressing LN229 cells than in mock-expressing LN229 cells. Consistent with these results, KML001 sensitized PTEN-deficient cells more strongly to growth inhibition than it did PTEN-positive cells in prostate and breast cancer cell lines. Finally, we illustrated in vivo anti-tumor effects of KML001 using an intracranial xenograft mouse model. These results suggest that KML001 could be an effective chemotherapeutic drug for the treatment of glioblastoma cancer patients with higher Akt activity and PTEN loss. Full article

Skeletal muscles are high-insulin tissues responsible for disposing of glucose via the highly regulated process of facilitated glucose transporter 4 (GLUT4). Impaired insulin action in diabetes, as well as disorders of GLUT4 vesicle trafficking in the muscle, are involved in defects in insulin-stimulated GLUT4 translocation. Since the Rab GTPases are the main regulators of vesicular membrane transport in exo- and endo-cytosis, in the present work, we studied the effect of olive leaf polyphenols (OLPs) on Rab8A, Rab13, and Rab14 proteins of the rat soleus muscle in a model of streptozotocin (SZT)-induced diabetes (DM) in a dose-dependent manner. Glucose, cholesterol, and triglyceride levels were determined in the blood, morphological changes of the muscle tissue were captured by hematoxylin and eosin histological staining, and expression of GLUT4, Rab8A, Rab13, and Rab14 proteins were analyzed in the rat soleus muscle by the immunofluorescence staining and immunoblotting. OLPs significantly reduced blood glucose level in all treated groups. Furthermore, significantly reduced blood triglycerides were found in the groups with the lowest and highest OLPs treatment. The dynamics of activation of Rab8A, Rab13, and Rab14 was OLPs dose-dependent and more effective at higher OLP doses. Thus, these results indicate a beneficial role of phenolic compounds from the olive leaf in the regulation of glucose homeostasis in the skeletal muscle. Full article

Methemoglobin (MetHb) is a hemoglobin (Hb) derivative with the heme iron in ferric state (Fe³⁺), unable to deliver oxygen. Quantification of methemoglobin is a very important diagnostic parameter in hypoxia. Recently, novel hemoglobin microparticles (Hb-MP) with a narrow size distribution around 700 nm, consisting of cross-linked Hb were proposed as artificial oxygen carriers. The cross-linking of Hb by glutaraldehyde (GA) generates a certain amount of MetHb. Due to the strong light scattering, quantitative determination of MetHb in Hb-MP suspensions by common spectrophotometry is not possible. Here, we demonstrate that ¹H₂O NMR relaxometry is a perfect tool for direct measurement of total Hb and MetHb concentrations in Hb-MP samples. The longitudinal relaxation rate 1/T₁ shows a linear increase with increasing MetHb concentration, whereas the transverse relaxation rate 1/T₂ linearly increases with the total Hb concentration. In both linear regressions the determination coefficient (R²) is higher than 0.99. The method does not require time-consuming pretreatment or digestion of the particles and is not impaired by light scattering. Therefore, it can be established as the method of choice for the quality control of Hb-MP and similar hemoglobin-based oxygen carriers in the future. Full article

Due to strong antimicrobial properties, silver nanoparticles (AgNPs) are used in a wide range of medical and consumer products, including those dedicated for infants and children. While AgNPs are known to exert neurotoxic effects, current knowledge concerning their impact on the developing brain is scarce. During investigations of mechanisms of neurotoxicity in immature rats, we studied the influence of AgNPs on glutamate transporter systems which are involved in regulation of extracellular concentration of glutamate, an excitotoxic amino acid, and compared it with positive control—Ag citrate. We identified significant deposition of AgNPs in brain tissue of exposed rats over the post-exposure time. Ultrastructural alterations in endoplasmic reticulum (ER) and Golgi complexes were observed in neurons of AgNP-exposed rats, which are characteristics of ER stress. These changes presumably underlie substantial long-lasting downregulation of neuronal glutamate transporter EAAC1, which was noted in AgNP-exposed rats. Conversely, the expression of astroglial glutamate transporters GLT-1 and GLAST was not affected by exposure to AgNPs, but the activity of the transporters was diminished. These results indicate that even low doses of AgNPs administered during an early stage of life create a substantial risk for health of immature organisms. Hence, the safety of AgNP-containing products for infants and children should be carefully considered. Full article

The strong association between diabetes mellitus type 2 and cancer is observed. The incidence of both diseases is increasing globally due to the interaction between them. Recent studies suggest that there is also an association between cancer incidence and anti-diabetic medications. An inhibitor of dipeptidyl-peptidase 4 (DPP-4), sitagliptin, is used in diabetes treatment. We examined the influence of sitagliptin alone or in combination with a cytostatic drug (paclitaxel) on the development of epithelial ovarian cancer cells and the process of metastasis. We examined migration, invasiveness, apoptosis, and metalloproteinases (MMPs) and their inhibitors’ (TIMPs) production in two human ovarian cancer cell lines. Sitagliptin induced apoptosis by caspase 3/7 activation in paclitaxel-treated SKOV-3 and OVCAR-3 cells. Sitagliptin maintained paclitaxel influence on ERK and Akt signaling pathways. Sitagliptin additionally reduced migration and invasiveness of SKOV-3 cells. There were distinct differences of metalloproteinases production in sitagliptin-stimulated ovarian cancer cells in both cell lines, despite their identical histological classification. Only the SKOV-3 cell line expressed MMPs and TIMPs. SKOV-3 cells co-treated with sitagliptin and paclitaxel decreased concentrations of MMP-1, MMP-2, MMP-7, MMP-10, TIMP-1, TIMP-2. The obtained data showed that sitagliptin used with paclitaxel may be considered as a possibility of pharmacological modulation of intracellular transmission pathways to improve the response to chemotherapy. Full article

Nucleophosmin (NPM1) gene mutations rarely occur in non-acute myeloid neoplasms (MNs) with <20% blasts. Among nearly 10,000 patients investigated so far, molecular analyses documented NPM1 mutations in around 2% of myelodysplastic syndrome (MDS) cases, mainly belonging to MDS with excess of blasts, and 3% of myelodysplastic/myeloproliferative neoplasm (MDS/MPN) cases, prevalently classified as chronic myelomonocytic leukemia. These uncommon malignancies are associated with an aggressive clinical course, relatively rapid progression to overt acute myeloid leukemia (AML) and poor survival outcomes, raising controversies on their classification as distinct clinico-pathologic entities. Furthermore, fit patients with NPM1-mutated MNs with <20% blasts could benefit most from upfront intensive chemotherapy for AML rather than from moderate intensity MDS-directed therapies, although no firm conclusion can currently be drawn on best therapeutic approaches, due to the limited available data, obtained from small and mainly retrospective series. Caution is also suggested in definitely diagnosing NPM1-mutated MNs with blast count <20%, since NPM1-mutated AML cases frequently present dysplastic features and multilineage bone marrow cells showing abnormal cytoplasmic NPM1 protein delocalization by immunohistochemical staining, therefore belonging to NPM1-mutated clone regardless of blast morphology. Further prospective studies are warranted to definitely assess whether NPM1 mutations may become sufficient to diagnose AML, irrespective of blast percentage. Full article

Autophagy refers to the process involving the decomposition of intracellular components via lysosomes. Autophagy plays an important role in maintaining and regulating cell homeostasis by degrading intracellular components and providing degradation products to cells. In vivo, autophagy has been shown to be involved in the starvation response, intracellular quality control, early development, and cell differentiation. Recent studies have revealed that autophagy dysfunction is implicated in neurodegenerative diseases and tumorigenesis. In addition to the discovery of certain disease-causing autophagy-related mutations and elucidation of the pathogenesis of conditions resulting from the abnormal degradation of selective autophagy substrates, the activation of autophagy is essential for prolonging life and suppressing aging. This article provides a comprehensive review of the role of autophagy in health, physiological function, and autophagy-related disease. Full article

The majority of the human proteome is subjected to N-terminal (Nt) acetylation catalysed by N-terminal acetyltransferases (NATs). The NatA complex is composed of two core subunits—the catalytic subunit NAA10 and the ribosomal anchor NAA15. Furthermore, NAA10 may also have catalytic and non-catalytic roles independent of NatA. Several inherited and de novo NAA10 variants have been associated with genetic disease in humans. In this study, we present a functional analysis of two de novo NAA10 variants, c.29A>G p.(D10G) and c.32T>G p.(L11R), previously identified in a male and a female, respectively. Both of these neighbouring amino acids are highly conserved in NAA10. Immunoprecipitation experiments revealed that both variants hamper complex formation with NAA15 and are thus likely to impair NatA-mediated Nt-acetylation in vivo. Despite their common impact on NatA formation, in vitro Nt-acetylation assays showed that the variants had opposing impacts on NAA10 catalytic activity. While NAA10 c.29A>G p.(D10G) exhibits normal intrinsic NatA activity and reduced monomeric NAA10 NAT activity, NAA10 c.32T>G p.(L11R) displays reduced NatA activity and normal NAA10 NAT activity. This study expands the scope of research into the functional consequences of NAA10 variants and underlines the importance of understanding the diverse cellular roles of NAA10 in disease mechanisms. Full article

The specific value of IgA Anti-β2glycoprotein I antibodies (aB2GP1) in the diagnosis and management of antiphospholipid syndrome (APS) is still controversial and a matter of active debate. The relevance of the IgA aB2GP1 isotype in the pathophysiology of APS has been increasingly studied in the last years. There is well know that subjects with multiple positive APS tests are at increased risk of thrombosis and/or miscarriage. However, these antibodies are not included in the 2006 APS classification criteria. Since 2010 the task force of the Galveston International Congress on APS recommends testing IgA aB2GP1 isotype in patients with APS clinical criteria in the absence of criteria antibodies. In this review, we summarize the molecular and clinical “state of the art” of the IgA aB2GP in the context of APS. We also discuss some of the characteristics that may help to evaluate the real value of the IgA aB2GP1 determination in basic research and clinical practice. The scientific community should be aware of the importance of clarifying the role of IgA aB2GP1 in the APS diagnosis. Full article

Cryptosporidiumparvum is a clinically important eukaryotic parasite that causes the disease cryptosporidiosis, which manifests with gastroenteritis-like symptoms. The protist has mitosomes, which are organelles of mitochondrial origin that have only been partially characterized. The genome encodes a highly reduced set of transport proteins of the SLC25 mitochondrial carrier family of unknown function. Here, we have studied the transport properties of one member of the C. parvum carrier family, demonstrating that it resembles the mitochondrial ADP/ATP carrier of eukaryotes. However, this carrier has a broader substrate specificity for nucleotides, transporting adenosine, thymidine, and uridine di- and triphosphates in contrast to its mitochondrial orthologues, which have a strict substrate specificity for ADP and ATP. Inspection of the putative translocation pathway highlights a cysteine residue, which is a serine in mitochondrial ADP/ATP carriers. When the serine residue is replaced by cysteine or larger hydrophobic residues in the yeast mitochondrial ADP/ATP carrier, the substrate specificity becomes broad, showing that this residue is important for nucleotide base selectivity in ADP/ATP carriers. Full article

Imatinib, one of the most used therapeutic agents to treat leukemia, is an inhibitor that specifically blocks the activity of tyrosine kinases. The molecule of imatinib is flexible and contains several functional groups able to take part in H-bonding and hydrophobic interactions. Analysis of molecular conformations for this drug was carried out using density functional theory calculations of rotation potentials along single bonds and by analyzing crystal structures of imatinib-containing compounds taken from the Cambridge Structural Database and the Protein Data Bank. Rotation along the N-C bond in the region of the amide group was found to be the reason for two relatively stable molecular conformations, an extended and a folded one. The role of various types of intermolecular interactions in stabilization of the particular molecular conformation was studied in terms of (i) the likelihood of H-bond formation, and (ii) their contribution to the Voronoi molecular surface. It is shown that experimentally observed hydrogen bonds are in accord with the likelihood of their formation. The number of H-bonds in ligand-receptor complexes surpasses that in imatinib salts due to the large number of donors and acceptors of H-bonding within the binding pocket of tyrosine kinases. Contribution of hydrophilic intermolecular interactions to the Voronoi molecular surface is similar for both conformations, while π...π stacking is more typical for the folded conformation of imatinib. Full article

Diabetic kidney disease (DKD) is the leading cause of chronic kidney disease and end-stage kidney disease. Renin–angiotensin system inhibitors such as losartan are the predominant therapeutic options in clinical practice to treat DKD. Therefore, it is necessary to identify DKD-related metabolic profiles that are affected by losartan. To investigate the change in metabolism associated with the development of DKD, we performed global and targeted metabolic profiling using 800 MHz nuclear magnetic resonance spectroscopy of urine samples from streptozotocin-induced diabetic mice (DM) with or without losartan administration. A principal component analysis plot showed that the metabolic pattern in the losartan-treated diabetic mice returned from that in the DM group toward that in the control mice (CM). We found that 33 urinary metabolites were significantly changed in DM compared with CM, and the levels of 16 metabolites among them, namely, glucose, mannose, myo-inositol, pyruvate, fumarate, 2-hydroxyglutarate, isobutyrate, glycine, threonine, dimethylglycine, methyldantoin, isoleucine, leucine, acetylcarnitine, 3-hydroxy-3-methylglutarate, and taurine, shifted closer to the control level in response to losartan treatment. Pathway analysis revealed that these metabolites were associated with branched-chain amino acid degradation; taurine and hypotaurine metabolism; glycine, serine, and threonine metabolism; the tricarboxylic acid cycle; and galactose metabolism. Our results demonstrate that metabolomic analysis is a useful tool for identifying the metabolic pathways related to the development of DKD affected by losartan administration and may contribute to the discovery of new therapeutic agents for DKD. Full article

Abnormal activation of Toll-like receptor (TLRs) signaling can result in colon cancer development. The aim of this study was to investigate the expression of important TLRs in different histological types of colorectal polyps and evaluate their relationship with intestinal microbiota. The expression levels of TLR2, 3, 4, and 5 were analyzed in intestinal biopsy specimens of 21 hyperplastic polyp (HP), 16 sessile serrated adenoma (SSA), 29 tubular adenoma (TA), 21 villous/tubulovillous (VP/TVP) cases, and 31 normal controls. In addition, selected gut bacteria including Streptococcus bovis, Enterococcus faecalis, Enterotoxigenic Bacteroides fragilis (ETBF), Fusobacterium nucleatum, Porphyromonas spp., Lactobacillus spp., Roseburia spp., and Bifidobacterium spp. were quantified in fecal samples using absolute qRT PCR, and, finally, the association between TLRs and these gut microbiota- was evaluated by Spearman’s correlation coefficient. Higher expression of TLR2 and TLR4 in VP/TVP and TA, and lower expression levels of TLR3 and TLR5 in all type of polyps were observed. The differences in TLR expression patterns was not only dependent on the histology, location, size, and dysplasia grade of polyps but also related to the intestinal microbiota patterns. TLR2 and TLR4 expression was directly associated with the F. nucleatum, E. faecalis, S. bovis, Porphyromonas, and inversely to Bifidobacterium, Lactobacillus, and Roseburia quantity. Furthermore, TLR3 and TLR5 expression was directly associated with Bifidobacterium, Roseburia, and Lactobacillus quantity. Our results suggest a possible critical role of TLRs during colorectal polyp progression. An abnormal regulation of TLRs in relation to gut microbial quantity may contribute to carcinogenesis. Full article

Inflammation is the main pathophysiology of dry eye, characterized by tear film instability and hyperosmolarity. The aim of this study was to investigate the association of inflammation and cellular autophagy using an in vitro dry eye model with primary cultured human corneal epithelial cells (HCECs). Primary HCECs cultured with fresh limbal explants from donors were switched to a hyperosmotic medium (450 mOsM) by adding sodium chloride into the culture medium. We observed the stimulated inflammatory mediators, TNF-α, IL-1β, IL-6 and IL-8, as well as the increased expression of autophagy related genes, Ulk1, Beclin1, Atg5 and LC3B, as evaluated by RT-qPCR and ELISA. The immunofluorescent staining of LC3B and Western blotting revealed the activated autophagosome formation and autophagic flux, as evidenced by the increased LC3B autophagic cells with activated Beclin1, Atg5, Atg7 and LC3B proteins, and the decreased levels of P62 protein in HCECs. Interestingly, the autophagy activation was later at 24 h than inflammation induced at 4 h in HCECs exposed to 450 mOsM. Furthermore, application of rapamycin enhanced autophagy activation also reduced the inflammatory mediators and restored cell viability in HCECs exposed to the hyperosmotic medium. Our findings for the first time demonstrate that the autophagy activation is a late phase response to hyperosmotic stress, and is enhanced by rapamycin, which protects HCECs by suppressing inflammation and promoting cells survival, suggesting a new therapeutic potential to treat dry eye diseases. Full article

Macrophages are mononuclear cells that become osteoclasts (OCs) in the presence of two cytokines, macrophage colony-stimulating factor (M-CSF), and receptor activator of NF-κB ligand (RANKL). RANKL binding to its specific receptor RANK leads to OCs differentiation mainly by nuclear factor of activated T-cells cytoplasmic 1 (NFATc1). In our previous study, the analysis of the protein network in NFATc1-knockdown cells, using the Ingenuity Pathway Analysis (IPA), showed a link between NFATc1 and Mitogen-activated protein kinase kinase (MEK)-extracellular receptor kinase (ERK) signaling pathway. Therefore, this study aimed to extend our knowledge of the relationship between NFATc1 and the ERK. Here, we demonstrate that delayed ERK1/2 phosphorylation in pre-OC RANKL-induced depends on NFATc1. Indeed, the knockdown of NFATc1 reduced the phosphorylation of ERK1/2 (60%) and the pharmacological inhibition of the ERK1/2 kinase activity impairs the expression of NFATc1 without preventing its translocation into the nucleus. Furthermore, silencing of NFATc1 significantly reduced RANKL-induced migration (p < 0.01), and most pre-OCs are still mononuclear after 48 h (80 ± 5%), despite the presence of actin rings. On the other hand, the inhibitors FR180204 and PD98059 significantly reduced RANKL-induced cell migration (p < 0.01), leading to a reduction in the number of multinucleated cells. Finally, we suggest that long-lasting ERK activity depends on NFATc1 induction and is likely linked to cell migration, fusion, and OC differentiation. Full article

The Brassica genus contains abundant economically important vegetable and oilseed crops, which are under threat of diseases caused by fungal, bacterial and viral pathogens. Resistance gene analogues (RGAs) are associated with quantitative and qualitative disease resistance and the identification of candidate RGAs associated with disease resistance is crucial for understanding the mechanism and management of diseases through breeding. The availability of Brassica genome assemblies has greatly facilitated reference-based quantitative trait loci (QTL) mapping for disease resistance. In addition, pangenomes, which characterise both core and variable genes, have been constructed for B. rapa, B. oleracea and B. napus. Genome-wide characterisation of RGAs using conserved domains and motifs in reference genomes and pangenomes reveals their clustered arrangements and presence of structural variations. Here, we comprehensively review RGA identification in important Brassica genome and pangenome assemblies. Comparison of the RGAs in QTL between resistant and susceptible individuals allows for efficient identification of candidate disease resistance genes. However, the reference-based QTL mapping and RGA candidate identification approach is restricted by the under-represented RGA diversity characterised in the limited number of Brassica assemblies. The species-wide repertoire of RGAs make up the pan-resistance gene analogue genome (pan-RGAome). Building a pan-RGAome, through either whole genome resequencing or resistance gene enrichment sequencing, would effectively capture RGA diversity, greatly expanding breeding resources that can be utilised for crop improvement. Full article

Canine malignant mammary gland tumors present with a poor prognosis due to metastasis to other organs, such as lung and lymph node metastases. Unlike in human studies where obesity has been shown to increase the risk of breast cancer, this has not been well studied in veterinary science. In our preliminary study, we discovered that leptin downregulated cathepsin A, which is responsible for lysosomal-associated membrane protein 2a (LAMP2a) degradation. LAMP2a is a rate-limiting factor in chaperone-mediated autophagy and is highly active in malignant cancers. Therefore, in this study, alterations in metastatic capacity through cathepsin A by leptin, which are secreted at high levels in the blood of obese patients, were investigated. We used a canine inflammatory mammary gland adenocarcinoma (CHMp) cell line cultured with RPMI-1640 and 10% fetal bovine serum. The samples were then subjected to real-time polymerase chain reaction, Western blot, immunocytochemistry, and lysosome isolation to investigate and visualize the metastasis and chaperone-mediated autophagy-related proteins. Results showed that leptin downregulated cathepsin A expression at both transcript and protein levels, whereas LAMP2a, the rate-limiting factor of chaperone-mediated autophagy, was upregulated by inhibition of LAMP2a degradation. Furthermore, leptin promoted LAMP2a multimerization through the lysosomal mTORC2 (mTOR complex 2)/PH domain and leucine rich repeat protein phosphatase 1 (PHLPP1)/AKT1 (Serine/threonine-protein kinase 1) pathway. These findings suggest that targeting leptin receptors can alleviate mammary gland cancer cell metastasis in dogs. Full article

MicroRNAs (miRNAs) are non-coding RNAs that regulate diverse cellular pathways by controlling gene expression. Increasing evidence has revealed their critical involvement in influenza A virus (IAV) pathogenesis. Host–IAV interactions induce different levels of oxidative stress (OS) by disrupting the balance between reactive oxygen species (ROS) and antioxidant factors. It is thought that miRNA may regulate the expression of ROS; conversely, ROS can induce or suppress miRNA expression during IAV infection. Thus, miRNA and OS are the two key factors of IAV infection and pathogenesis. Accordingly, interactions between OS and miRNA during IAV infection might be a critical area for further research. In this review, we discuss the crosstalk between miRNAs and OS during IAV infection. Additionally, we highlight the potential of miRNAs as diagnostic markers and therapeutic targets for IAV infections. This knowledge will help us to study host–virus interactions with novel intervention strategies. Full article