Targeted image-guided oncologic surgery (IGOS) relies on the recognition of cell surface-associated proteins, which should be abundantly present on the tumor cells but preferably absent on cells in surrounding healthy tissue. The transmembrane receptor tyrosine kinase EphA2, a member of the A class of the Eph receptor family, has been reported to be highly overexpressed in several tumor types including breast, lung, brain, prostate, and colon cancer, and is considered amongst the most promising cell membrane-associated tumor antigens by the NIH. Another member of the Eph receptor family belonging to the B class, EphB4, has also been found to be up-regulated in multiple cancer types. In this study, EphaA2 and EphB4 are evaluated as target for IGOS of colorectal cancer by immunohistochemistry (IHC) using a tissue microarray (TMA) consisting of 168 pairs of tumor and normal tissue. The IHC sections were scored for staining intensity and percentage of cells stained. The results show a significantly enhanced staining intensity and more widespread distribution in tumor tissue compared with adjacent normal tissue for EphA2 as well as EphB4. Based on its more consistently higher score in colorectal tumor tissue compared to normal tissue, EphB4 appears to be an especially promising candidate for IGOS of colorectal cancer.

Fasudil, a potent Rho kinase (ROCK) inhibitor, can ameliorate LPS-induced acute lung injury (ALI) in mice, but the mechanism remains obscure. In this study, a mice model of ALI was established by intra-tracheal instillation of LPS. Histological changes, cytokine levels, lung permeability, and endothelial apoptosis were determined to evaluate the effects of fasudil on lung injury. The cellular and molecular biological mechanisms were explored by culturing human pulmonary microvascular endothelial cells (PMECs). The results showed that fasudil reduced LPS-induced lung inflammation, pulmonary hyperpermeability, and endothelial apoptosis in mice. In cultured human PMECs, fasudil inhibited LPS-induced caspse-3 cleavage and cell apoptosis. It also decreased LPS-induced hyperpermeability of human PMECs monolayer by reversing the down-regulation of intercellular junctions. Moreover, fasudil inhibited LPS-induced overexpression of chemokines and intercellular adhesion molecule (ICAM)-1 in human PMECs, which in turn suppressed neutrophil chemotaxis and neutrophil-endothelial adhesion. Further molecular researches showed fasudil inhibited LPS-induced activation of ROCK, NF-κB, and p38 in human PMECs. Our findings demonstrated that fasudil alleviated LPS-induced ALI by protecting endothelial function via inhibiting endothelial apoptosis, maintaining endothelial barrier integrity, and reducing endothelial inflammation. These effects of fasudil could be attributed to the inhibition of ROCK and its downstream NF-κB and p38 signaling pathways.

The epidermal growth factor receptor (EGFR) plays vital roles in cellular processes including cell proliferation, survival, motility and differentiation. Dysregulated activation of the receptor is often implicated in human cancers. EGFR is synthesized as a single-pass transmembrane protein, which consists of an extracellular ligand-binding domain and an intracellular kinase domain separated by a single transmembrane domain. The receptor is activated by a variety of polypeptide ligands such as epidermal growth factor and transforming growth factor α. It has long been thought that EGFR is activated by ligand-induced dimerization of the receptor monomer, which brings intracellular kinase domains into close proximity for trans-autophosphorylation. An increasing number of diverse studies, however, demonstrate that EGFR is present as a pre-formed, yet inactive, dimer prior to ligand binding. Furthermore, recent progress in structural studies has provided insight into conformational changes during the activation of a pre-formed EGFR dimer. Upon ligand binding to the extracellular domain of EGFR, its transmembrane domains rotate or twist parallel to the plane of the cell membrane, resulting in reorientation of the intracellular kinase domain dimer from a symmetric inactive configuration to an asymmetric active form (the “rotation model”). This model is also able to explain how oncogenic mutations activate the receptor in the absence of ligand without assuming that the mutations induce receptor dimerization. In this review, we discuss mechanisms underlying ligand-induced activation of the preformed EGFR dimer, as well as how oncogenic mutations constitutively activate the receptor dimer, based on the rotation model.

Regulated autophagy is involved in the repair of renal ischemia-reperfusion injury (IRI). ω3-Polyunsaturated fatty acids (ω3-PUFAs) show protective effects against various renal injuries. It was recently reported that ω3-PUFAs regulate autophagy. We assessed whether ω3-PUFAs attenuated IR-induced acute kidney injury (AKI) and evaluated associated mechanisms. C57Bl/6 background fat-1 mice and wild-type mice (wt) were divided into four groups: wt sham (n = 10), fat-1 sham (n = 10), wt IRI (reperfusion 35 min after clamping both the renal artery and vein; n = 15), and fat-1 IRI (n = 15). Kidneys and blood were harvested 24 h after IRI. Renal histological and molecular data were collected. The kidneys of fat-1 mice showed better renal cell survival, renal function, and pathological damage than those of wt mice after IRI. In addition, fat-1 mice showed less oxidative stress and autophagy impairment; greater amounts of LC3, Beclin-1, and Atg7; lower amounts of p62; and higher levels of renal cathepsin D and ATP6E than wt kidneys. They also showed more AMPK activation, which resulted in the inhibition of phosphorylation of the mammalian target of rapamycin (mTOR). Collectively, ω3-PUFAs in fat-1 mice contributed to AMPK mediated autophagy activation, leading to a renoprotective response.

Abnormality of fibroblast growth factor receptors (FGFRs) mediated signaling pathways were frequently found in various human malignancies, making FGFRs hot targets for cancer treatment. To address the consistent need of new chemotype of FGFR inhibitors, here, we started with a hit structure identified from our internal c-Met inhibitor project, and conducted a chemical optimization. After exploring three parts of the hit compound, we finally discovered a new series of pyrrolo[2,3-b] pyrazine FGFR inhibitors, which contain a novel scaffold and unique molecular shape. We believed that our findings can benefit others to further develop selective FGFR inhibitors.

Pyruvate kinase M2 (PKM2) is essential for aerobic glycolysis and is highly expressed in various cancer tissues. Although high PKM2 expression is observed in prostate cancer tissues, its functional role in cancer metabolism is unclear. Here, we investigated the role of PKM2 in regulating autophagy and its associated pathways in prostate cancer cells. PKM2 expression was silenced using various PKM2 small interfering RNAs (siRNAs) and then we measured PKM2-related cellular pathways associated with autophagy. PKM2 siRNA-transfected prostate cancer cells showed significantly reduced viability. Acridine orange staining and immunoblotting analysis showed that PKM2 downregulation markedly increased autophagic cell death. Results of western blotting analysis showed that PKM2 knockdown affected protein kinase B/mechanistic target of rapamycin 1 pathway, which consequently downregulated the expression of glycolytic enzymes lactate dehydrogenase A and glucose transporter 1. To the best of our knowledge, this is the first study to show that PKM2 inhibition alters cancer cell metabolism and induces autophagy. Thus, the present study provides a strategy for the development of PKM2-targeted novel anticancer drugs for the treatment of prostate cancer.

Aurora kinase A (AURKA) is a normal cell proliferation-inducing enzyme encoded by AURKA gene, with over-expression observed in different types of malignancies. Hence, the goal is to find potential inhibitors against AURKA. In this study, molecular docking, Standard Precision and Extra Precision methods were employed. After the docking study, the ligands showed an extremely low binding score which suggested very high binding affinity of the ligands. Furthermore, Quantum polarized ligand docking (QPLD) was performed to predict the binding status of the molecules. Based on the binding affinity, the top four compounds were chosen for further analysis. The docked complexes were further analyzed in explicit water conditions using 100 ns molecular dynamics simulations and binding free energy calculation. Then, density functional theory (DFT) calculation was used to calculate the molecular properties of the molecules. Finally, systems biology experiments validated the molecular docking and molecular dynamics simulation studies and indicated that quercetin, kaempferol, luteolin and rutin could inhibit the AURKA. The results show that, these four molecules have high binding affinity to the AURKA and significant interactions (LEU139, GLU211and ALA213) were also identified with the hinge region of Aurora kinase A. Thus, LEU139, GLU211, and ALA213 were identified as the crucial protein mechanisms.

The search for improved therapeutic approaches to breast cancer are still on going. Breast tumor kinase (BRK, also known as PTK6) is one of the targets, it is highly expressed in breast carcinomas while displaying low or no expression in the normal mammary gland, which hints at the oncogenic role of this enzyme in breast cancer. In these twenty years, an increasing number of studies have focused on understanding the cellular roles of BRK in breast cancers. This review outlines the advances made towards understanding the cellular and physiological role of BRK, the molecular and chemical inhibitors and its therapeutic significance in breast cancer.

Populations with essential hypertension have a high risk of nonalcoholic steatohepatitis (NASH). In this study, we investigated the mechanism that underlies the progression of hypertension-associated NASH by comparing differences in the development of high fat and cholesterol (HFC) diet-induced NASH among three strains of rats, i.e., two hypertensive strains comprising spontaneously hypertensive rats and the stroke-prone spontaneously hypertensive 5/Dmcr, and the original Wistar Kyoto rats as the normotensive control. We investigated histopathological changes and molecular signals related to inflammation in the liver after feeding with the HFC diet for 8 weeks. The diet induced severe lobular inflammation and fibrosis in the livers of the hypertensive rats, whereas it only caused mild steatohepatitis in the normotensive rats. Increased activation of proinflammatory signaling (transforming growth factor-β1/mitogen-activated protein kinases pathway) was observed in the hypertensive strains fed with the HFC diet. In addition, the HFC diet suppressed the nuclear factor erythroid 2-related factor 2 pathway in the hypertensive rats and led to lower increases in the hepatic expression of heme oxygenase-1, which has anti-oxidative and anti-inflammatory activities. In conclusion, these signaling pathways might play crucial roles in the development of hypertension-associated NASH.

The enzyme-coupled transient receptor potential channel subfamily M member 7, TRPM7, has been associated with immunity and immune cell signalling. Here, we review the role of this remarkable signalling protein in lymphocyte proliferation, differentiation, activation and survival. We also discuss its role in mast cell, neutrophil and macrophage function and highlight the potential of TRPM7 to regulate immune system homeostasis. Further, we shed light on how the cellular signalling cascades involving TRPM7 channel and/or kinase activity culminate in pathologies as diverse as allergic hypersensitivity, arterial thrombosis, and graft versus host disease (GVHD), stressing the need for TRPM7 specific pharmacological modulators.

Ataxin-2 (human gene symbol ATXN2, protein ATXN2) is the disease protein of Spinocerebellar Ataxia type 2 (SCA2). The large expansions of a polyglutamine (polyQ) stretch above a threshold of ~33 glutamines cause the multi-system nervous atrophy SCA2, while intermediate expansions of 29-32 glutamines contribute to the risk of the motor neuron diseases Amyotrophic Lateral Sclerosis (ALS) and Fronto-Temporal Lobar Dementia (FTLD). To elucidate the cellular function of ATXN2, we further characterized its direct interaction with alpha-Actinin-1 (symbol ACTN1), which had been observed in high-throughput yeast-two-hybrid surveys. An endogenous complex of ATXN2 and ACTN1 proteins was detected by co-immunoprecipitation. In vitro GST-tag pull-down experiments showed that the Calponin-Homology-domain at the N-terminus of ACTN1 binds to the N-terminus of ATXN2. Although an impact of the polyQ expansion on the interaction was not evident in pull-down experiments, a recent characterization of aged Atxn2-CAG100-KnockIn mice provides evidence. Both proteins associated in the cytosol and at the plasma membrane, as determined by sedimentation experiments in mouse brain, and by immunofluorescence microscopy of a transfected monkey cell line and of rat primary hippocampal neurons. In view of the roles of ACTN1 for spine plasticity and postsynaptic receptor control via reassembly of cortical actin, our data help to explain the impaired dendrite maintenance in SCA2.

Objective: The Rho GTPase/Rho kinase pathway is an important target in glaucoma treatment. This study investigated the hypotensive effect of RKI-1447, a Rho kinase inhibitor developed for cancer treatment, in a porcine ex vivo pigmentary glaucoma model. Materials and Methods: Twenty-eight fresh porcine anterior chambers were perfused with pigment medium (1.67 × 10⁷ pigment particles/mL) for 48 hours before being subjected to the RKI-1447 (n = 16) or the vehicle control (n = 12). Another twelve eyes with normal medium perfusion served as the control. The intraocular pressure (IOP) was recorded at two-minute intervals and the outflow facility was calculated. To investigate the intracellular mechanism of the IOP reduction, primary trabecular meshwork cells were exposed to RKI-1447 or the vehicle control and then analyzed for changes in cytoskeleton, motility, and phagocytosis. Results: Compared to the baseline, the perfusion of pigment caused a significant increase in IOP in the RKI-1447 group (P = 0.003) at 48 hours. Subsequent treatment with RKI-1447 significantly reduced IOP from 20.14 ± 2.59 mmHg to 13.38 ± 0.91 mmHg (P = 0.02). Pigment perfusion reduced the outflow facility from 0.27 ± 0.03 at baseline to 0.18 ± 0.02 at 48 hours (P < 0.001). This was partially reversed with RKI-1447. RKI-1447 exhibited no apparent changes in the micro- or macroscopic appearance, including histology. Primary TM cells exposed to RKI-1447 showed a significant disruption of the actin cytoskeleton both in the presence and absence of pigment exposure (P < 0.001) but no effect on TM migration was observed. Pigment-treated TM cells exhibited a reduction in TM phagocytosis, which RKI reversed. Conclusions: RKI-1447 is a novel ROCK inhibitor that significantly reduces IOP by disrupting TM stress fibers and increasing TM phagocytosis. These features may make it especially useful for the treatment of secondary glaucomas with an increased phagocytosis load but also for other open angle glaucomas.

Parkinson’s disease (PD) is characterized by motor deficits and a wide variety of non-motor symptoms. The age of onset, rate of disease progression and the precise profile of motor and non-motor symptoms display considerable individual variation. Neuropathologically, the loss of substantia nigra dopaminergic neurons is a key feature of PD. The vast majority of PD patients exhibit alpha-synuclein aggregates in several brain regions, but there is also great variability in the neuropathology between individuals. While the dopamine replacement therapies can reduce motor symptoms, current therapies do not modify the disease progression. Numerous clinical trials using a wide variety of approaches have failed to achieve disease modification. It has been suggested that the heterogeneity of PD is a major contributing factor to the failure of disease modification trials, and that it is unlikely that a single treatment will be effective in all patients. Precision medicine, using drugs designed to target the pathophysiology in a manner that is specific to each individual with PD, has been suggested as a way forward. PD patients can be stratified according to whether they carry one of the risk variants associated with elevated PD risk. In this review we assess current clinical trials targeting two enzymes, leucine-rich repeat kinase 2 (LRRK2) and glucocerebrosidase (GBA), which are encoded by two most common PD risk genes. Because the details of the pathogenic processes coupled to the different LRRK2and GBA risk variants are not fully understood, we ask if these precision medicine-based intervention strategies will prove “precise“ or “personalized“ enough to modify the disease process in PD patients. We also consider at what phases of the disease that such strategies might be effective, in light of the genes being primarily associated with the risk of developing disease in the first place, and less clearly linked to the rate of disease progression. Finally, we critically evaluate the notion that therapies targeting LRRK2 and GBA might be relevant to a wider segment of PD patients, beyond those that actually carry risk variants of these genes.

Purpose: This study investigated the hypotensive effect of RKI-1447, a Rho kinase inhibitor, in a porcine ex vivo pigmentary glaucoma model. Methods: Twenty-eight porcine anterior chambers were perfused with medium supplemented with 1.67 × 10⁷ pigment particles/mL for 48 hours before treatment with RKI-1447 (n = 16) or vehicle control (n = 12). Intraocular pressure (IOP) was recorded and outflow facility was calculated. Primary trabecular meshwork cells were exposed to RKI-1447 or vehicle control; effects on the cytoskeleton, motility, and phagocytosis were evaluated. Result: Compared to baseline, the perfusion of pigment caused a significant increase in IOP in the RKI-1447 group (P = 0.003) at 48 hours. Subsequent treatment with RKI-1447 significantly reduced IOP from 20.14 ± 2.59 mmHg to 13.38 ± 0.91 mmHg (P = 0.02). Pigment perfusion reduced the outflow facility from 0.27 ± 0.03 at baseline to 0.18 ± 0.02 at 48 hours (P < 0.001). This was partially reversed with RKI-1447. RKI-1447 caused no apparent histological changes in the micro- or macroscopic TM appearance. RKI-1447-treated primary TM cells showed significant disruption of the actin cytoskeleton both in the presence and absence of pigment (P < 0.001) but no effect on TM migration was observed. Pigment-treated TM cells exhibited a reduction in TM phagocytosis, which RKI-1447 reversed. Conclusion: RKI-1447 significantly reduces IOP by disrupting TM stress fibers and increasing TM phagocytosis. These features may make it useful for the treatment of secondary glaucomas with an increased phagocytic load.

Ovarian cancer is the common cause of death among gynecological cancers. Although ovarian cancer initially responds to chemotherapy, the frequent recurrence in patients remains a therapeutic challenge. Pyruvate kinase M2 (PKM2) plays a pivotal role in regulating cancer cell survival. However, its therapeutic roles remain unclear. Here, we investigated the anticancer effects of compound 3K, a specific PKM2 inhibitor, on autophagic and apoptotic pathway regulation in SK-OV-3 (PKM2-overexpressing human ovarian adenocarcinoma cell line). The anticancer effect of compound 3K was examined using the MTT and colony formation assay in SK-OV-3. The results of tissue microarray showed that PKM2 expression positively correlated with the severity of the tumor. Moreover, the expression of pro-apoptotic proteins increased in SK-OV-3 following compound 3K treatment. Compound 3K induced AMPK activation, which was accompanied by the inhibition of mTOR. Additionally, this compound inhibited glycolysis, resulting in reduced proliferation in SK-OV-3. Compound 3K treatment suppressed tumor progression in vivo xenograft model. Our findings suggest that the inhibition of PKM2 by compound 3K affected Warburg effects and induced autophagic cell death. Therefore, the use of specific PKM2 inhibitors to block the glycolytic pathway and target cancer cell metabolism represents a promising therapeutic approach for treating PKM2-overexpressing ovarian cancer.

Akt1 is a proto-oncogene that is over active in most cancers. Akt1 activation requires phosphorylation at Thr308; phosphorylation at Ser473 further enhances catalytic activity. Akt1 activity is also regulated via interactions between the kinase domain and the N-terminal auto-inhibitory pleckstrin homology (PH) domain. As it was previously difficult to produce Akt1 in site-specifically phosphorylated forms, the contribution of each activating phosphorylation site to auto-inhibition was unknown. Using a combination of genetic code expansion and in vivo enzymatic phosphorylation, we produced Akt1 variants containing programmed phosphorylation to probe the interplay between Akt1 phosphorylation status and the auto-inhibitory function of the PH domain. Deletion of the PH domain increased the enzyme activity for all three phosphorylated Akt1 variants. For the doubly phosphorylated enzyme, deletion of the PH domain relieved auto-inhibition by 295-fold. We next found that phosphorylation at Ser473 provided resistance to chemical inhibition by Akti-1/2 inhibitor VIII. The Akti-1/2 inhibitor was most effective against pAkt1T308 and showed 4-fold decreased potency with Akt1 variants phosphorylated at Ser473. The data highlight the need to design more potent Akt1 inhibitors that are effective against the doubly phosphorylated and most pathogenic form of Akt1.

Identification of recurrent driver mutations in genes encoding tyrosine kinases has resulted in the development of molecularly targeted strategies designed to improve the outcomes for patients diagnosed with acute myeloid leukemia (AML). The receptor tyrosine kinase FLT3, is the most commonly mutated gene in AML, with internal tandem duplications within the juxtamembrane domain (FLT3-ITD) or missense mutations in the tyrosine kinase domain (FLT3-TKD), present in 30%-35% of AML patients at diagnosis. An established driver mutation and marker of poor prognosis, the FLT3 tyrosine kinase has emerged as an attractive therapeutic target, and thus has encouraged the development of FLT3 tyrosine kinase inhibitors (TKIs). However, the therapeutic benefit of FLT3 inhibition, particularly as monotherapy, frequently results in the development of treatment resistance and disease relapse. Commonly, FLT3 inhibitor resistance is induced by the emergence of secondary lesions in the FLT3 gene, particularly in the second tyrosine kinase domain at residue Asp835 (D835) to form a ‘dual mutation’ (ITD-D835). Individual FLT3-ITD and FLT3-TKD mutations influence independent signaling cascades however, currently little is known which divergent signaling pathways are controlled by each of these FLT3 specific mutations, particularly in the context of patients harboring dual ITD-D835 mutations. This review provides a comprehensive analysis of the known discrete and cooperative signaling pathways regulated by each of the FLT3 specific mutations, as well as the therapeutic approaches that hold the most promise for development of more durable and personalized therapeutic approaches targeting mutant FLT3, to improve the treatment of AML.

Neuroinflammatory responses are implicated in the pathogenesis of neurodegenerative diseases. In neurodegenerative diseases, neuroinflammatory reactions to neuronal injury are modulated by microglial cells, which are vital innate immune cells in the central nervous system. Activated microglial cells release proinflammatory cytokines, mediators, and neurotoxic factors that induce fatal neuronal injury. The present study investigated the anti-neuroinflammatory effects of cudratricusxanthone L (1), which was isolated from Cudrania tricuspidata. This compound reduced the levels of lipopolysaccharide-stimulated inflammatory mediators and cytokines, including nitric oxide, prostaglandin E2, interleukin (IL)-1β, tumor necrosis factor-α, IL-6, and IL-12. These effects suggested that cudratricusxanthone L (1) suppressed the nuclear factor-kappa B (NF-κB) signaling pathway. Specifically, cudratricusxanthone L (1) also attenuated the phosphorylation of Jun kinase and inhibited p38 mitogen-activated protein kinase (MAPK) signaling in BV2 and rat primary microglial cells. These results indicated that cudratricusxanthone L (1) effectively repressed neuroinflammatory processes in BV2 and rat primary microglial cells by inhibiting NF-κB and the MAPK signaling pathway.

The transforming growth factor-β (TGF-β), in which overexpression have been associated with various diseases, has become an attractive molecular target for the treatment of cancers. Three series of 3-substituted-4-(quinoxalin-6-yl) pyrazoles 14a–h, 15a–h, 16a–h, 22a, 22b, 22d, 23a, 23b, 23d, 24b, and 24d were synthesized and evaluated for their activin receptor-like kinase 5 (ALK5) and p38α mitogen activated protein (MAP) kinase inhibitory activity in an enzymatic assays. Among these compounds, the most active compound 16f inhibited ALK5 phosphorylation with an IC₅₀ value of 0.28 µM, with 98% inhibition at 10 µM. Compound 16f also had good selectivity index of >35 against p38α MAP kinase, with 9.0-fold more selective than clinical candidate, compound 3 (LY-2157299). Molecular docking study was performed to identify the mechanism of action of the synthesized compounds and their good binding interactions were observed. ADMET prediction of good active compounds showed that these ones possess good pharmacokinetics and drug-likeness behavior.

Despite the impact of immune checkpoint inhibitors on malignancies treatment is unprecedented, a lack of response to these molecules is observed in several cases. Differently from melanoma and non-small cell lung cancer, where the use of immune checkpoint inhibitors results in a high efficacy, the response rate in other tumors, such as gastrointestinal cancers, breast cancer, sarcomas, and part of genitor-urinary cancers remains low. The first strategy evaluated to improve the response rate to immune checkpoint inhibitors is the use of predictive factors for the response as PD-L1 expression, tumor mutational burden, and clinical features. In addition to the identification of the patients with a high sensibility to immune checkpoint inhibitors, another approach currently under intensive investigation is the use of therapeutics in a combinatory manner with immune checkpoint inhibitors to obtain an enhancement of efficacy through the modification of the tumor immune microenvironment. In addition to the abscopal effect induced by radiotherapy, a lot of studies are evaluating several drugs able to improve response rate to immune checkpoint inhibitors, including microbiota modifiers, drugs targeting co-inhibitors receptors, anti-angiogenic therapeutics, small molecules, and oncolytic viruses. In view of the rapid and extensive development of this research field, we conducted a systematic review of the literature identifying which of these drugs are closer to achieving validation in the clinical practice.

Breast Cancer (BC) is the second most common type of cancer worldwide and displays the highest cancer-related mortality among women worldwide. Targeted therapies have revolutionized the way BC has been treated in the last decades improving life expectancies of millions of women. Among the different molecular pathways that have been of interest for the development of targeted therapies are the Cyclin-Dependent Kinases (CDK). CDK inhibitors are a class of molecules that already exist in nature and those belonging to the INK4 protein family specifically inhibit the CDK4/6 proteins. CDK4/6 inhibitors specifically block the transition from the G1 to the S phase of the cell cycle by dephosphorylation of the retinoblastoma tumor suppressor protein. In the past four years CDK4/6 inhibitors palbociclib, ribociclib, and abemaciclib received their first FDA approval for the treatment of Hormone Receptor (HR)-positive and Human Epidermal growth factor Receptor 2 (HER2)-negative breast cancer after showing significant improvements in progression-free survival in the PALOMA-1, MONALEESA-2 and the MONARCH-2 randomized clinical trials, respectively. After the encouraging results from these clinical trials, CDK4/6 inhibitors have also been investigated in the other BC subtypes. In HER2-positive BC, combination of CDK4/6 inhibitors with HER2-targeted therapies showed promise in preclinical studies and their clinical evaluation is ongoing. Moreover, in triple-negative BC, CDK4/6 inhibitors efficacy has been investigated in combination with other targeted therapies or immunotherapies. This review summarizes the molecular background and clinical efficacy of CDK4/6 inhibitors as single agents or in combination with other targeted therapies for the treatment of BC. Future directions of ongoing clinical trials and predictive biomarkers will be further debated.

Desmoplastic small round cell tumor (DSRCT) is an extremely rare, aggressive sarcoma affecting adolescents and young adults with male predominance. Generally, it originates from serosal surface of abdominal cavity. The hallmark characteristic of DSRCT is the EWSR1-WT1 gene fusion. This translocation up-regulates the expression of PDGFRα, VEGF and other proteins related to tumor and vascular cell proliferation. Current management of DSRCT includes a combination of chemotherapy, radiation and aggressive cytoreductive surgery plus intra-peritoneal hyperthermic chemotherapy (HIPEC). Despite advances in multimodal therapy, outcomes remain poor since the majority of patients present disease recurrence and die within 3 years. The dismal survival makes DSRCT an orphan disease with urgent need of new drugs. The treatment of advanced and recurrent disease with tyrosine kinase inhibitors, such as pazopanib, sunitinib, and mTOR inhibitors have been evaluated in small studies. Recent works using comprehensive molecular profiling of DSRCT identified potential therapeutic targets. In this review, we aim to describe the current studies conducted to better understand DSRCT biology and to explore the new therapeutic strategies under investigation in preclinical models and in early phase clinical trials.

Pancreatic ductal adenocarcinoma (PDA) remains the most lethal malignancy due to lack of an effective treatment. P21-activated kinases (PAKs) play key roles in PDA growth, and the PAK inhibitor PF-3758309 synergistically reduced PDA growth with gemcitabine. The aim of this study was to determine the effect of PF-3758309 with multiple chemotherapeutic reagents on a panel of patient-derived PDA cell lines. Cells were treated with PF-3758309 plus or minus gemcitabine, 5-fluorouracil (5-FU) or abraxane, and cell proliferation was determined. Protein expression profiles were measured by Western blot. PDA cells were subcutaneously injected into the flanks of SCID mice which were then treated with PF-3758309, gemcitabine, PF-3758309 plus gemcitabine, or gemcitabine plus abraxane. Tumour growth was measured by volume and weight. PF-3758309 enhanced the inhibitory effects of 5-FU, gemcitabine and abraxane on a panel of patient-derived PDA cells, inhibited HIF-1 protein expression and reduced the protein levels of palladin and -SMA in these cells. The combination of PF-3758309 with gemcitabine maximally inhibited PDA growth in vivo, which was comparable to the combination of gemcitabine with abraxane. PF-3758309 enhanced the suppressive effects of multiple chemotherapeutic reagents on the growth of a panel of patient-derived PDA cell lines. The combination of PF-3758309 with gemcitabine provides a potential treatment option with less toxicity than gemcitabine plus abraxane.

Background: In the heart, aldosterone (Aldo) binds the mineralocorticoid receptor (MR) to exert damaging, adverse remodeling-promoting effects. We recently showed that G protein-coupled receptor (GPCR)-kinase (GRK)-5 blocks the cardiac MR by directly phosphorylating it, thereby repressing its transcriptional activity. MR antagonist (MRA) drugs block the cardiac MR reducing morbidity and mortality of advanced human heart failure. Non-steroidal MRAs, such as finerenone, may provide better cardio-protection against Aldo than classic, steroidal MRAs, like spironolactone and eplerenone. Herein, we sought to investigate potential differences between finerenone and eplerenone at engaging GRK5-dependent cardiac MR phosphorylation and subsequent blockade. Methods: We used the cardiomyocyte cell line H9c2 and neonatal rat ventricular myocytes (NRVMs). Results: GRK5 phosphorylates the MR in H9c2 cardiomyocytes in response to finerenone but not to eplerenone. Unlike eplerenone, finerenone alone potently and efficiently suppresses cardiac MR transcriptional activity, thus displaying inverse agonism. GRK5 is necessary for finerenone`s inverse agonism, since GRK5 genetic deletion renders finerenone incapable of blocking cardiac MR transcriptional activity. Eplerenone alone does not fully suppress cardiac MR basal activity regardless of GRK5 expression levels. Finally in NRVMs, GRK5 is necessary for the anti-apoptotic and anti-fibrotic effects of both finerenone and eplerenone against Aldo, as well as for the higher efficacy and potency of finerenone at blocking Aldo-induced apoptosis and fibrosis. Conclusions: Finerenone, but not eplerenone, induces GRK5-dependent cardiac MR inhibition, which underlies, at least in part, its higher potency and efficacy, compared to eplerenone, as an MRA in the heart. GRK5 acts as a co-repressor of the cardiac MR and is essential for efficient MR antagonism in the myocardium.

Congenital anomalies of the kidney and urinary tract (CAKUT) are common birth defects deriving from abnormalities in renal differentiation during embryogenesis. CAKUT is the major cause of end-stage renal disease and chronic kidney diseases in children, but its genetic causes remain largely unresolved. Here we discuss advances in the understanding of how MAPK/ERK activity contributes to the regulation of ureteric bud branching morphogenesis, which dictates the final size, shape, and nephron number of the kidney. Recent studies also demonstrate that MAPK/ERK pathway is directly involved in nephrogenesis, regulating both the maintenance and differentiation of the nephrogenic mesenchyme. Interestingly, aberrant MAPK/ERK signaling is linked to many cancers, and recent studies suggest it also plays a role in the most common pediatric renal cancer, Wilms’ tumor.

The mineralocorticoid hormone aldosterone regulates sodium and potassium homeostasis but also adversely modulates the maladaptive process of cardiac adverse remodeling post-myocardial infarction. Through activation of its mineralocorticoid receptor (MR), a classic steroid hormone receptor/transcription factor, aldosterone promotes inflammation and fibrosis of the heart, the vasculature, and the kidneys. This is why MR antagonists reduce morbidity and mortality of heart disease patients and are part of the mainstay pharmacotherapy of advanced human heart failure. A plethora of animal studies using cell type–specific targeting of the MR gene have established the importance of MR signaling and function in cardiac myocytes, vascular endothelial and smooth muscle cells, renal cells, and macrophages. In terms of its signaling properties, the MR is distinct from nuclear receptors in that it has, in reality, two physiological hormonal agonists: not only aldosterone but also cortisol. In fact, in several tissues, including in the myocardium, cortisol is the primary hormone activating the MR. There is a considerable amount of evidence indicating that the effects of the MR in each tissue expressing it depend on tissue- and ligand-specific engagement of molecular co-regulators that either activate or suppress its transcriptional activity. Identification of these co-regulators for every ligand that interacts with the MR in the heart (and in other tissues) is of utmost importance therapeutically, since it can not only help elucidate fully the pathophysiological ramifications of the cardiac MR`s actions but also help design and develop novel better MR antagonist drugs for heart disease therapy. Among the various proteins the MR interacts with are molecules involved in cardiac G protein-coupled receptor (GPCR) signaling. This results in a significant amount of crosstalk between GPCRs and the MR, which can affect the latter`s activity dramatically in the heart and in other cardiovascular tissues. This review summarizes the current experimental evidence for this GPCR-MR crosstalk in the heart and discusses its pathophysiological implications for cardiac adverse remodeling as well as for heart disease therapy. Novel findings revealing non-conventional roles of GPCR signaling molecules, specifically of GPCR-kinase (GRK)-5, in cardiac MR regulation are also highlighted.

Rett syndrome (RTT), a neurodevelopmental disorder, is mainly caused by mutations in methyl CpG-binding protein 2 (MECP2), which alter the functions of domains to either bind to methylated DNA or interact with a transcriptional co-repressor complex. It has been established that alterations in cyclin-dependent kinase-like 5 (CDKL5) or forkhead box protein G1 (FOXG1) correspond to distinct neurodevelopmental disorders, given that a series of studies have indicated that RTT is also caused by alterations in either one of these genes. We tried to elucidate RTT through evolution and structure assessment of MeCP2, CDKL5, and FOXG1, by focusing on their binding partners and disordered structures. Here, we provide insight into the similarities of the FOXG1 and MECP2 binding partners evolution and function. On the other hand, we suggest that CDKL5 could be a potential candidate for a classical RTT treatment, particularly based on its disordered structure that spans after the catalytic domain to the C-terminus, which shows abundant linear motifs that can bind to molecules with divergent structures of similar affinity. Additionally, we provide insight into the relationship between disordered structure and disease.

Prototype of a family of at least nine members, c-src tyrosine kinase is a therapeutically interesting target, because its inhibition might be of interest not only in a number of malignancies, but also in a diverse array of conditions, from neurodegenerative pathologies to certain viral infections. Computational methods in drug discovery are considerably cheaper than conventional methods and offer opportunities of screening very large numbers of compounds in conditions that would be simply impossible within the wet lab experimental settings. We have explored the use of global QSAR models and molecular ligand docking in the discovery of new c-src tyrosine kinase inhibitors. Using a data set of 1038 compounds from ChEMBL and 19 blocks of molecular descriptors, we have developed over 200 QSAR classification models, based on six machine learning algorithms and 17 feature selection methods. We have selected 49 with reasonably good performance (positive predictive value and balanced accuracy higher than 70% in nested cross validation) and the models were assembled by stacking with a simple majority vote and used for the virtual screening of over the “named” ZINC data set (over 100,000 compounds). 744 compounds were predicted by at least 50% of the QSAR models as active, 147 compounds were within the applicability domain and predicted by at least 75% of the models to be active. The latter 147 compounds were submitted to molecular ligand docking using Vina and Ledock, and a number of 90 were predicted to be active based on the binding energy. External data from CHEMBL and PUBCHEM confirmed that at least 7.83% (in the case of QSAR) or 6.67% (in the case of integrated QSAR and molecular docking) of the compounds are active on the c-src target.

The use of the Bruton’s tyrosine kinase (BTK) inhibitors has changed the management and clinical history of patients with chronic lymphocytic leukemia (CLL). BTK is a critical molecule that interconnects B-cell antigen receptor (BCR) signaling. BTKIs are classified into two categories: irreversible (covalent) inhibitors and reversible (non-covalent) inhibitors. Ibrutinib is the first irreversible BTK inhibitor approved by the U.S. Food and Drug Administration in 2013 as a breakthrough therapy in CLL patients. Subsequently, several studies evaluated the efficacy and safety of new agents with reduced toxicity when compared with ibrutinib. Two other irreversible, second-generation BTK inhibitors, acalabrutinib and zanubrutinib, were developed to reduce ibrutinib-mediated adverse effects. Additionally, new reversible BTK inhibitors are currently under development in an early phase studies to improve their activity and to diminish adverse effects. This review summarizes the pharmacology, clinical efficacy, safety, dosing, drug-drug interactions associated with the treatment of CLL with BTK inhibitors, and examines its further implications.

COVID-19 has been characterized as one of the deadliest respiratory diseases, and the emergence of SARS-CoV-2 caught the pharmaceutical industry and the drug development communities off guard. Identifying potential antiviral targets is of great concern, and one way to detect them is by analyzing metabolic changes in infected cells. In this study, we present a novel workflow to predict robust druggable targets against emerging RNA viruses using metabolic networks and information of the viral structure and its genome sequence. For this purpose, we implemented pymCADRE, a tool to create metabolic models using gene expression data, and used this to reconstruct a metabolic network of the human bronchial epithelial cells. We observed that pymCADRE reduces the computational time when flux variability analysis is employed for internal optimizations. Subsequently, we created a fully automated computational tool, named PREDICATE, which analyses one or more nucleotide sequences, introduces given amino acid mutations, and simulates in silico viral infections. Moreover, it predicts a set of host reactions, which, when constrained, inhibit the virus production while preserving the host’s optimal state. In the context of SARS-CoV-2, we applied this tool to our metabolic network of bronchial epithelial cells and identified enzymatic reactions with inhibitory effects. From the list of the reported targets, the most promising one was the Nucleoside Diphosphate Kinase, whose inhibitors have already been reported in the literature. Finally, we computationally tested the robustness of our targets in all currently known variants of concern, verifying the inhibitory effect of our target enzyme against SARS-CoV-2. Focusing on the metabolic fluxes of infected cells, we aim at applying our workflow and methods for rapid hypothesis-driven identification of potentially exploitable antivirals to efficiently prevent future pandemics concerning various viruses and host cell types.

This randomized trial compared pea protein, whey protein, and water-only supplementation on muscle damage, inflammation, delayed onset of muscle soreness (DOMS), and physical fitness test performance during a 5-day period after a 90-minute eccentric exercise bout in non-athletic, non-obese males (n=92, ages 18-55 years). The two protein sources (0.9 g protein/kg divided into three doses/day) were administered under double blind procedures. The eccentric exercise protocol induced significant muscle damage and soreness, and reduced bench press and 30-second Wingate performance. Whey protein supplementation significantly attenuated post-exercise blood levels for biomarkers of muscle damage compared to water-only, with large effect sizes for creatine kinase and myoglobin during the 4th and 5th days of recovery (Cohen's d &gt;0.80); pea protein versus water supplementation had an intermediate, non-significant effect (Cohen's d &lt;0.50); and no significant differences between whey and pea protein were found. Whey and pea protein compared to water supplementation had no significant effects on post-exercise DOMS and the fitness tests. In conclusion, high intake of whey protein for 5 days after intensive eccentric exercise mitigated efflux of muscle damage biomarkers, with intake of pea protein having an intermediate effect in part due to the 24% lower leucine amino acid content.

The MAP kinase ERK5 contains an N-terminal kinase domain and a unique C-terminal tail including a nuclear localization signal and a transcriptional activation domain. ERK5 is activated in response to growth factors and stresses, and regulates transcription at the nucleus by either phosphorylation or interaction with transcription factors. MEK5-ERK5 pathway plays an important role regulating cancer cell proliferation and survival. Therefore, it is important to define the precise molecular mechanisms implicated in ERK5 nucleo-cytoplasmic shuttling. We previously described that the molecular chaperone Hsp90 stabilizes and anchors ERK5 at the cytosol, and that ERK5 nuclear shuttling requires Hsp90 dissociation. Here, we show that MEK5 or Cdc37 overexpression -mechanisms that induce nuclear ERK5- induced ERK5 SUMO-2 modification at residues Lys6/Lys22 in cancer cells. We also show that overexpression of the SUMO protease SENP2 completely abolished endogenous ERK5 nuclear localization in response to EGF stimulation. Furthermore, mutation of these SUMO sites abolished the ability of ERK5 to translocate to the nucleus and to promote prostatic cancer PC-3 cell proliferation. These results allow us to propose a more precise mechanism: in response to MEK5 activation, ERK5 SUMOylation favors the dissociation of Hsp90 from the complex, allowing ERK5 nuclear shuttling and activation of transcription.

The purpose of this work is to investigate the protein kinase inhibitory activity of constituents from ethyl acetate soluble fraction of Acacia auriculiformis stem bark. Column chromatography, gel filtration and NMR spectroscopy were used to purified and characterized betulin from the extract. Betulin which is a known inducer of apoptosis was screened against a panel of 16 disease-related protein kinases. Betulin was shown to inhibit Abelson murine leukemia viral oncogene homolog 1 (ABL1) kinase, casein kinase 1epsilon (CK1epsilon), glycogen synthase kinase 3alpha/β (GSK-3alpha/β), Janus kinase 3 (JAK3), NIMA Related Kinase 6 (NEK6) and vascular endothelial growth factor receptor 2 kinase (VEGFR2) and with activity in µM range. The effect of betulin on the cell viability of doxorubicin-resistant K562R chronic myelogenous leukemia cells was then verified to underline its putative use as anti-cancer compound. Betulin was shown to modulate the mitogen-activated protein (MAP) kinase pathway similarly to imatinib mesylate, a well-known inhibitor of ABL1 kinase. The interaction of betulin and ABL1 was studied by molecular docking showing an interaction of the inhibitor with the ATP binding pocket. Altogether, these data demonstrate that betulin is a multi-target inhibitor of protein kinases, an activity that can contribute to the anticancer properties of the natural compound and notably for treatment of leukemia.

The involvement of astrocytic SN1 (SNAT3) transporter in ammonia-induced L-glutamine retention was recently documented in mouse cultured astrocytes. Here we investigated the involvement of specificity protein 1 (Sp1) transcription factor in SN1 regulation in ammonium chloride (“ammonia”)-treated astrocytes. Sp1 expression and its cellular localization were determined using real-time qPCR, Western blot and confocal microscopy, respectively. Sp1 binding to Snat3 promoter was analyzed by chromatin immunoprecipitation. Ammonia-induced Sp1 regulatory role in SN1-mediated [³H]glutamine transport was verified using siRNA and mithramycin A. The involvement of protein kinase C (PKC) isoforms in Sp1 level/phosphorylation status was verified using siRNA technology. Sp1 translocation to the nuclei and its enhanced binding to Snat3 promoter, along with Sp1 dependence of system N-mediated [³H]glutamine transport were observed in astrocytes upon ammonia exposure. Ammonia decreased the level of phosphorylated Sp1, and the effect was reinforced by long-term incubation with PKC modulator, phorbol 12-myristate 13-acetate, a treatment likely to dephosphorylate Sp1. Furthermore, silencing of PKCδ isoform abolished the increase of Sp1 level by ammonia. Collectively, the results demonstrate the regulatory role of Sp1 in regulation of SN1 expression and activity in ammonia-treated astrocytes and implicate altered Sp1 phosphorylation status in this capacity.

The mammalian/mechanistic target of rapamycin complex 1 (mTORC1) is activated by the small G-protein, RHEB-GTPase. On lysosome, RHEB activates mTORC1 by binding the domains of N-heat, M-heat, and FAT, which allosterically regulates ATP binding in the active site for further phosphorylation. The crucial role of RHEB in regulating growth and survival through mTORC1, makes it a targetable site for anti-cancer therapeutics. However, the binding kinetics of RHEB to mTORC1 is still unknown at the molecular level. Therefore, we studied the kinetics by in vitro and in-cell protein-protein interaction (PPI) assays. For this, we used the split-luciferase system (NanoBiT®) for in-cell studies, and prepared proteins for the in vitro measurements. Consequently, it was shown that RHEB binds to the whole mTOR both in the presence or absence of GTPɣS, with five-fold weaker affinity in the presence of GTPɣS. Also, RHEB bound to the truncated mTOR fragments of N-heat domain (60-167) and M-heat domain (967-1023) in a GTP independent manner. Furthermore, RHEB bound to the truncated kinase domain (2148-2300) with higher affinity also in GTP independently. In conclusion, RHEB binds two different binding sites of mTOR, which probably regulates the kinase activity of mTOR through multiple different molecular mechanisms.

Rett syndrome (RTT) is mainly caused by mutations in methyl CpG-binding protein 2, cyclin-dependent kinase-like 5, or forkhead box protein G1. These RTT-causing proteins harbor an intrinsically disordered region (IDR) whose conformation exhibits spatiotemporal heterogeneity, which not only confer versatility to the protein, but also implicates them in diseases. The IDR generally evolves more rapidly than an ordered structure. In this study, we examined the relationship between pathogenic RTT-associated point mutations in RTT-causing proteins and the evolutionary dynamics of sequence features including structural order–disorder, phosphorylation sites, and evolutionary rates. We also analyzed the molecular properties and evolution of proteins that interact with RTT-causing proteins in terms of phylogenetic profiles, tissue specificity, subcellular localization, expression level, and functions. The results indicate that constrained IDRs may function by forming contacts with other regions in the protein sequence causing pathogenic missense mutations likely to arise in the rapidly evolving IDR and affect molecular networks, leading to disease. The results also provide novel insights into the genetic basis for RTT and the evolution of the neocortex in higher vertebrates.

Hypothesis: Anti-diabetic drugs modulate p-21 activated kinase (PAK) signaling Introduction: Type 2 diabetes mellitus (T2DM) is a chronic inflammatory disease associated with increased cancer risk. PAK signaling is implicated in cellular homeostasis when regulated, and cancer when unrestrained. Recent reports provided a role for PAK signaling in glucose homeostasis, but the role of PAKs in the pathogenesis of T2DM is unknown. Here we explored whether PAK signaling should be targeted via chemoprevention to reduce diabetes-associated cancer risk. Methods: PAK interacting partners in T2DM were identified using online STRING database. Systematic literature review provided the effect of anti-diabetic drugs on PAK signaling. Review of clinical studies revealed the overall clinical response rate and percentage of adverse events in piogliazone (n=53) and metformin (n=91) treated patients with PAK-dependent diseases. Results: A total of 30 PAK interacting partners were identified (10: reduced beta-cell mass; 10: beta-cell dysfunction; 10: obesity-insulin resistance) which were highly associated with Wnt, and G-protein signaling. Anti-diabetic drug metformin activated signaling pathways upstream; whereas pioglitazone inhibited pathways downstream of PAK. Overall clinical response upon pioglitazone treatment was 53%. 79% of pioglitazone and 75% of metformin treated patients had adverse events. Pioglitazone reduced molecular-PAK biomarkers of proliferation (Ki67 and CyclinD1), and metformin had the opposite effect. Conclusions: PAK signaling in T2DM involves Wnt and G-protein signaling which is altered by anti-diabetic drugs metformin and pioglitazone. Apart from the therapeutic limitations of adverse events, pioglitazone is promising in chemoprevention, however long-term multi-centered studies, which initiate pioglitazone treatment early will be required to fully assess the full potential of these drugs.

The ascomycete Trichoderma atroviride is well known for its mycoparasitic lifestyle. Similar to other organisms, light is an important cue for T. atroviride. However, besides triggering of conidiation, little is known on the physiological responses of T. atroviride to light. In this study, we analyzed how cultivation under different light wavelengths and regimes impacted the behavior of two T. atroviride wild-type strains, IMI206040 and P1. While colony extension of both strains was slightly affected by light, massive differences in the photoconidation response between the two strains became evident. T. atroviride P1 colonies conidiated under all conditions tested including growth in complete darkness, while IMI206040 required white, blue or green light to trigger asexual reproduction. Interestingly, deletion of the stress-activated MAP kinase-encoding gene tmk3 abolished the ability of strain P1 to conidiate in red and yellow light as well as in darkness. Furthermore, light-dependent differences in the mycoparasitic activity of T. atroviride and in the biosynthesis of the secondary metabolite 6-pentyl--pyrone (6-PP) became evident. 6-PP production was highest upon dark incubation while light, especially exposure to white light as light/dark cycles, had an inhibitory effect on its biosynthesis. We conclude that the response of T. atroviride to light is strain-dependent and impacts differentiation, mycoparasitism and 6-PP production and hence should be considered in experiments testing the mycoparasitic activity of these fungi.

The proliferation and survival signals emanating from the B-cell receptor (BCR) constitute a crucial aspect of mature lymphocyte’s life. Dysregulated BCR signaling is considered a potent contributor to tumor survival in different subtypes of B cell non-Hodgkin lymphomas (B-NHLs). In the last decade, emergence of BCR-associated kinases as rational therapeutic targets has led to the development and approval of several small molecule inhibitors targeting either Bruton's tyrosine kinase (BTK), spleen tyrosine kinase (SYK), or phosphatidylinositol 3 kinase (PI3K), offering alternative treatment options to standard chemoimmunotherapy, and making some of these drugs valuable assets in the anti-lymphoma armamentarium. Despite their initial effectiveness, these precision medicine strategies are limited by primary resistance in aggressive B-cell lymphoma like diffuse large B cell lymphoma (DLBCL) and mantle cell lymphoma (MCL), especially in the case of first generation BTK inhibitors. In these patients, BCR-targeting drugs often fail to produce durable responses, and nearly all cases eventually progress with a dismal outcome, due to secondary resistance. This review will discuss our current understanding of the role of antigen-dependent and antigen-independent BCR signaling in DLBCL and MCL and will cover both approved inhibitors and investigational molecules being evaluated in early preclinical studies. We will discuss how the mechanisms of action of these molecules, and their off/on-target effects can influence their effectiveness and lead to toxicity, and how our actual knowledge supports the development of more specific inhibitors and new, rationally based, combination therapies, for the management of MCL and DLBCL patients.

The group III two-component hybrid histidine kinase MoHik1p in the filamentous fungus Magnaporthe oryzae is known to be a sensor for external osmotic stress and essential for the fungicidal activity of the phenylpyrrole fludioxonil. The mode of action of fludioxonil has not yet been completely clarified but rather assumed to hyperactivate the high osmolarity glycerol (HOG) signaling pathway. To date, not much is known about the detailed molecular mechanism of how osmotic stress is detected or fungicidal activity is initiated within the HOG pathway. The molecular mechanism of signaling was studied using a mutant strain in which the HisKA signaling domain was modified by an amino acid change of histidine H736 to alanine A736. We found that MoHik1pH736A is as resistant to fludioxonil but not as sensitive to osmotic stress as the null mutant ∆Mohik1. H736 is required for fludioxonil action but is not essential for sensing sorbitol stress. Consequently, this report provides evidence of the difference in the molecular mechanism of fludioxonil action and the perception of osmotic stress. This is an excellent basis to understand the successful phenylpyrrole-fungicides’ mode of action better and will give new ideas to decipher cellular signaling mechanisms.

Hypothesis: Anti-diabetic drugs modulate p-21 activated kinase (PAK) signaling Introduction: Type 2 diabetes mellitus (T2DM) is a chronic inflammatory disease associated with increased cancer risk. PAK signaling is implicated in cellular homeostasis when regulated, and cancer when unrestrained. Recent reports provided a role for PAK signaling in glucose homeostasis, but the role of PAKs in the pathogenesis of T2DM is unknown. Here we performed a mini-meta analysis to explore if anti-diabetic drugs modify PAK signaling pathways, and provide insight regarding modulation of these pathways to potentially reduce diabetes-associated cancer risk. Methods: PAK interacting partners in T2DM were identified using online STRING database. Correlation studies were performed via systematic literature review to understand the effect of anti-diabetic drugs on PAK signaling. Mini meta analysis correlated multiple clinical studies and revealed the overall clinical response rate and percentage of adverse events in piogliazone (n=53) and metformin (n=91) treated patients with PAK-associated diseases. Results: A total of 30 PAK interacting partners were identified (10: reduced beta-cell mass; 10: beta-cell dysfunction; 10: obesity-insulin resistance) which were highly associated with Wnt, and G-protein signaling. Anti-diabetic drug metformin activated signaling pathways upstream; whereas pioglitazone inhibited pathways downstream of PAK. Overall clinical response upon pioglitazone treatment was 53%. 79% of pioglitazone and 75% of metformin treated patients had adverse events. Pioglitazone reduced molecular-PAK biomarkers of proliferation (Ki67 and CyclinD1), and metformin had the opposite effect. Conclusions: PAK signaling in T2DM likely involves Wnt and G-protein signaling which may be altered by anti-diabetic drugs metformin and pioglitazone. Apart from the therapeutic limitations of adverse events, pioglitazone may be promising in chemoprevention, however long-term multi-centered studies, which initiate pioglitazone treatment early will be required to fully assess the full potential of these drugs.

Genetic activation of the class I PI3K pathway is very common in cancer. This mostly results from oncogenic mutations in PIK3CA, the gene encoding the ubiquitously expressed PI3Kα catalytic subunit, or from inactivation of the PTEN tumour suppressor, a lipid phosphatase which opposes class I PI3K signalling. The clinical impact of PI3K inhibitors in solid tumours, aimed at dampening cancer-cell-intrinsic PI3K activity, has thus far been limited. Challenges include poor drug tolerance, incomplete pathway inhibition and pre-existing or inhibitor-induced resistance. The principle of pharmacologically targeting cancer-cell-intrinsic PI3K activity also assumes that all cancer-promoting effects of PI3K activation are reversible, which might not be the case. Emerging evidence suggests that genetic PI3K pathway activation can induce and/or allow cells to tolerate chromosomal instability, which – even if occurring in a low fraction of the cell population – might help to facilitate and/or drive tumour evolution. While it is clear that such genomic events cannot be reverted pharmacologically, a role for PI3K in the regulation of chromosomal instability could be exploited by using PI3K pathway inhibitors to prevent those genomic events from happening and/or reduce the pace at which they are occurring, thereby dampening cancer development or progression. Such an impact might be most effective in tumours with clonal PI3K activation and achievable at lower drug doses than the maximum-tolerated doses of PI3K inhibitors currently used in the clinic.

Fusarium wilt of banana is a devastating disease that has decimated banana production worldwide. Host resistance to Fusarium oxysporum f. sp. cubense (Foc), the causal agent of this disease, is genetically dissected in this study using two Musa acuminata ssp. malaccensis segregating populations segregating for Foc Tropical (TR4) and Subtropical (STR4) race 4 resistance. Marker loci and trait association using 11 SNP-based PCR markers allowed the candidate region to be delimited to a 12.9 cM genetic interval corresponding to a 959 kb region on Chromosome 3 of ‘DH-Pahang’ reference assembly v4. Within this region, there is a cluster of pattern recognition receptors, namely leucine rich repeat ectodomain containing receptor-like protein kinases, cysteine-rich cell wall associated protein kinases, and leaf rust 10 disease-resistance locus receptor-like proteins positioned in an interspersed arrangement. Their transcript levels were rapidly upregulated in the resistant but not in susceptible F2 progenies at the onset of infection. This suggests that one or several of these genes may control resistance at this locus. To confirm the segregation of single-gene resistance, we generated an inter-cross between the resistant parent ‘Ma850’ and a susceptible line ‘Ma848’, to show that the STR4 resistance co-segregated with marker ‘28820’ at this locus. Finally, an informative SNP marker 29730 allowed the locus specific resistance to be assessed in a collection of diploid and polyploid banana plants. Out of the 60 lines screened, 22 lines were predicted to carry resistance at this locus, including lines known to be TR4 resistant, such as ‘Pahang’, ‘SH-3362’, ‘SH-3217’, ‘Ma-ITC0250’, and ‘DH-Pahang/CIRAD 930’. Additional screening in the International Institute for Tropical Agriculture’s collection suggests that the dominant allele is common among in the elite ‘Matooke’ NARITA hybrids, as well as in other triploid or tetraploid hybrids derived from East African highland bananas. Fine-mapping and candidate gene identification will allow characterization of molecular mechanisms underlying TR4 resistance. The markers developed in this study can now aid the marker-assisted selection of TR4 resistance in breeding programs around the world.

Chronic myeloid leukemia (CML) is a myeloproliferative neoplasm constituting approximately 15% of newly diagnosed leukemia in adult patients. Development of tyrosine kinase inhibitors (TKIs) have dramatically improved outcomes in patients with chronic CML in chronic phase. However, adverse drug events (ADEs) associated with TKI therapy have influenced drug adherence, resulting in adverse clinical outcomes and a decline in the quality of life (QoL). In this study, we carried out a unique questionnaire survey to evaluate ADEs, which comprised 14 adverse events. We compared drug adherence rates between patients using imatinib and those who switched from imatinib to nilotinib, a second-generation TKI. Following the switch, the total number of ADEs decreased considerably in most cases. Simultaneously, better QoL was observed in the nilotinib group than in the imatinib group. Drug adherence was measured using Morisky’s 9-item Medication Adherence Scale (MMAS). MMAS increased significantly after switching to nilotinib in all cases. Drug adherence is a critical factor for achieving molecular response in patients with CML. In fact, our results showed a strong inverse correlation between clinical outcome [international scale (IS)] and adherence (MMAS), with a stronger tendency in the nilotinib group than in the imatinib group. In conclusion, low occurrence of ADEs induced a high level of QoL and a good clinical response with second-generation TKI nilotinib treatment.

Objective: To investigate the protein expression levels of cyclin‑dependent kinase subunit 2 (CKS2) and cluster of differentiation (CD) 47 in gastric cancer (GC) and their clinical significances. Methods: A total of 126 GC patients who underwent radical resection were selected as study subjects. Additionally, 32 patients with benign gastric tumor, 42 patients with low-grade intraepithelial neoplasia (LGIEN), and 49 patients with high-grade intraepithelial neoplasia (HGIEN) who underwent surgery were selected as the control groups. Immunohistochemistry was used to detect the expression of CKS2 and CD47 in surgical specimens. We statistically analyzed the clinical significance of the expression of the two factors. Results: (1) The positivity rates for CKS2 in benign gastric tumor tissue, LGIEN tissue, HGIEN tissue, and GC tissue gradually increased, i.e., 6.3% (2/32), 30.9% (13/42), 38.8% (19/49), and 60.3% (76/126), respectively, and the positivity rates for CD47 were 18.8% (6/32), 38.1% (16/42), 46.9% (23/49), and 65.9% (83/126), respectively. (2) High expression of CKS2 and CD47 were associated with tumor diameter, Lauren classification, number of lymph node metastases, and TNM stage. In addition, the immunohistochemical scores for CKS2 and CD47 were positively correlated (r=0.625, P=0.000). (3) The median follow-up time of 126 patients was 46.5 months, and the overall survival rate was 40.5% (51/126). Survival analysis showed that compared with that in the CKS2 (-) group, the overall survival rate for patients in the CKS2 (+) group was significantly worse (25.0% vs 64.0%, 2=15.67, P=0.000) and that compared with the CD47 (-) group, the CD47 (+) group had significantly worse overall survival (30.1% vs 60.5%, 2=15.67, P=0.000). (4) The overall survival rates of CKS2(+)CD47(+) group, CKS2(+)CD47(-) group, CKS2(-)CD47(+) group, and CKS2 (-)CD47 (-) group were 20.0%(13/65), 58.3%(7/12), 57.1%(8/14), 65.7% (23/35), respectively, the prognosis of patients in CKS2(+)CD47(+) group was significantly poor. Conclusion: High expression levels of CKS2 and CD47 were closely related to the occurrence of GC and can be used as independent risk factors to assess the prognosis of patients.

Epithelial-to-mesenchymal transition (EMT) plays a role in chronic obstructive pulmonary diseases (COPD). Cyclic adenosine monophosphate (cAMP) can inhibit transforming growth factor-β1 (TGF-β1) mediated EMT. Although compartmentalization via A-kinase anchoring proteins (AKAPs) is central to cAMP signaling, functional studies on their therapeutic value in the lung EMT process are lacking. Bronchial epithelial (BEAS-2B, primary HAE cells) were exposed to TGF-β1. Epithelial (E-cadherin, ZO-1) and mesenchymal markers collagen Ӏ (mRNA, protein) were analyzed. St-Ht31 disrupted AKAP-PKA interactions. TGF-β1 release was measured by ELISA. TGF-β1-sensitive AKAPs Ezrin, AKAP95 and Yotiao were silenced using siRNA. Cell migration was analyzed by wound healing assay, xCELLigence, Incucyte. Prior to TGF-β1, dibutyryl-cAMP (dbcAMP), fenoterol, rolipram, cilostamide, forskolin were used to elevate intracellular cAMP. TGF-β1 induced morphological changes, decreased E-cadherin but increased collagen Ӏ and cell migration, a process reversed by PF-670462. TGF-β1 altered (mRNA, protein) expression of Ezrin, AKAP95 and Yotiao. St-Ht31 decreased E-cadherin (mRNA, protein), but counteracted TGF-β1-induced collagen Ӏ upregulation. Cigarette smoke (CS) increased TGF-β1 release, activated TGF signaling, augmented cell migration and reduced E-cadherin expression, a process blocked by TGF-β1 neutralizing antibody. Silencing of Ezrin, AKAP95 and Yotiao diminished TGF-β1-induced collagen Ӏ expression, as well as TGF-β1-induced cell migration. Fenoterol, rolipram, and cilostamide, in AKAP silenced cells pointed to distinct cAMP compartments. We conclude that Ezrin, AKAP95 and Yotiao promote TGF-β1-mediated EMT, linked to a TGF-β1 release by CS. AKAP members define the ability of fenoterol, rolipram and cilostamide to modulate the EMT process, and are potential relevant targets in the treatment of COPD.

Background: Real-world data on the molecular epidemiology of EGFR resistance mutations at or after progression with first- or second-generation EGFR-TKIs in patients with advanced NSCLC are lacking. Methods: This ongoing observational study was carried out by 23 hospital-based physicians in Greece. The decision to perform Cobas® EGFR Mutation Test v2 in tissue and/or plasma at disease progression was made before enrollment. For patients with negative/inconclusive T790M plasma-based results, tissue re-biopsy could be performed. Results: Ninety-six (96) eligible patients were consecutively enrolled (median age: 67.8 years) between July-2017 and September-2019. Of the patients, 98% were tested upon progression using plasma and 2% using tissue/cytology biopsy. The T790M mutation was detected in 16.0% of liquid biopsies. Tissue re-biopsy was performed in 22.8% of patients with a T790M-negative plasma result. In total, the T790M positivity rate was 21.9%, not differing between patients on first- or second-generation EGFR-TKI. Higher (≥2) ECOG performance status and longer (≥10 months) time to disease progression following EGFR-TKI treatment initiation were associated with T790M positivity. Conclusions: Results from plasma/tissue-cytology samples in a real-world setting, yielded a T790M positivity rate lower than previous reports. Fewer than one in four patients with negative plasma-based testing underwent tissue re-biopsy, indicating the challenges in routine care settings.

Manzamines are complex polycyclic marine-derived β-carboline alkaloids with reported anticancer, immunostimulatory, anti-inflammatory, antibacterial, antiviral, antimalarial, neuritogenic, hyperlipidemia and atherosclerosis suppression bioactivities, putatively associated with inhibition of glycogen synthase kinase-3, cyclin-dependent kinase 5, and vacuolar ATPases. We hypothesized that additional and yet undiscovered molecular targets might be associated with Manzamine A (MZA) reported pharmacological properties. We report herein for the first time to our knowledge that MZA inhibited a 90kDa ribosomal protein kinase S6 (RSK1) when screened against a panel of 30 protein kinases. Furthermore in vitro RSK kinase assays demonstrated a 10-fold selectivity in potency of MZA against RSK1 versus RSK2. MZA’s differential binding and selectivity toward the two isoforms is also supported by computational docking experiments. Specifically, the RSK1-MZA (N- and C-termini) complexes appear to have stronger interactions and preferable energetics contrary to the RSK2-MZA ones. In addition, our computational strategy suggests that MZA binds to the N-terminal kinase domain of RSK1 rather than the C-terminal domain. RSK is a vertebrate family of cytosolic serine-threonine kinases that act downstream of the ras-ERK1/2 (extracellular-signal-regulated kinase 1/2) pathway, which phosphorylates substrates shown to regulate several cellular processes including growth, survival and proliferation. Consequently, our findings have lead us to hypothesize that MZA and the 80 currently known manzamine-type alkaloids isolated from several sponge genera, may have novel pharmacological properties.

In most of acute myeloid leukemia patients there is an aberrant tyrosine kinases activity. The Sprouty family proteins were originally identified in Drosophila melanogaster as antagonists of Breathless, the mammalian ortholog of fibroblast growth factor receptor. This family proteins are inhibitors of RAS signaling induced by tyrosine kinases receptors and they are implicated in negative feedback processes regulating several intracellular pathways. The present study aims to investigate the role of a member of the Sprouty family, Sprouty1, as regulator of cell proliferation and growth in patients affected by acute myeloid leukemia. Sprouty1 mRNA and protein were both significantly down-regulated in acute myeloid leukemia cells compared to the normal counterpart, but they were restored when remission is achieved after chemotherapy. Ectopic expression of Sprouty1 revealed that it plays a key role in the proliferation and apoptotic defect that represent a landmark of the leukemic cells. Our study identified Sprouty1 as negative regulator involved in the aberrant signals of acute myeloid leukemia. Furthermore, we found a correlation between Sprouty1 and FoxO3a delocalization in AML at diagnosis, suggesting a multistep regulation of RAF–MEK–ERK signaling in human cancers.

Abstract: Oncogenic RAS mutations drive more than half of human cancers, and RAS inhibition is the holy grail of oncology. Thirty years of relentless efforts and harsh disappointments have taught us about the intricacies of oncogenic RAS signalling that allow us to now get a pharmacological grip on this elusive protein. The inhibition of effector pathways, such as the RAF-MEK-ERK pathway, has largely proven disappointing. So far, most of these efforts were aimed at blocking the activation of ERK. Here, we discuss RAF dependent pathways that are regulated through RAF functions independent of catalytic activity and their potential role as targets to block oncogenic RAS signalling. We focus on the now well documented roles of RAF kinase independent functions in apoptosis, cell cycle progression and cell migration.

Preproenkephalin (PPE) is a precursor molecule for multiple endogenous opioid peptides Leu-enkephalin (ENK) and Met-ENK, which are involved in a wide variety of modulatory functions in the nervous system. Despite the functional importance of ENK in the brain, the effect of brain-derived factor(s) on PPE expression is unknown. We report the dual effect of neural epidermal growth factor (EGF)-like-like 2 (NELL2) on PPE gene expression. In cultured NIH3T3 cells, transfection of NELL2 expression vectors induced an inhibition of PPE transcription intracellularly, in parallel with downregulation of protein kinase C signaling pathways and extracellular signal-regulated kinase. Interestingly, these phenomena were reversed when synthetic NELL2 was administered extracellularly. The in vivo disruption of NELL2 synthesis resulted in an increase in PPE mRNA level in the rat brain, suggesting that the inhibitory action of intracellular NELL2 predominates the activation effect of extracellular NELL2 on PPE gene expression in the brain. Biochemical and molecular studies with mutant NELL2 structures further demonstrated the critical role of EGF-like repeat domains in NELL2 for regulation of PPE transcription. These are the first results to reveal the spatio-specific role of NELL2 in the homeostatic regulation of PPE gene expression.

Huntington’s Disease (HD) is a devastating neurodegenerative disorder caused by a CAG trinucleotide repeat expansion in the HTT gene, for which no disease modifying therapies are currently available. Much of the recent research has focused on developing therapies to directly lower HTT expression, and while promising, these therapies have presented several challenges regarding administration and efficacy. Another promising therapeutic approach is the modulation of HTT post-translational modifications (PTMs) that are dysregulated in disease and have shown to play a key role in HTT toxicity. Among all PTMs, modulation of HTT phosphorylation has been proposed as an attractive therapeutic option due to the possibility of orally administering specific kinase effectors. One of the kinases described to participate in HTT phosphorylation is Protein Kinase CK2. CK2 has recently emerged as a target for the treatment of several neurological and psychiatric disorders, although its role in HD remains controversial. While pharmacological studies in vitro inhibiting CK2 resulted in reduced HTT phosphorylation and increased toxicity, genetic approaches in mouse models of HD have provided beneficial effects. In this review we discuss potential therapeutic approaches related to the manipulation of HTT-PTMs with special emphasis on the role of CK2 as a therapeutic target in HD.

Nanoprecipitation by liquid anti-solvent precipitation is one of the most versatile methods to produce pure drug nanoparticles (PDNPs) owing to the ability to optimize the properties of the product. Nevertheless, nanoprecipitation shows broad particle size distribution and low physical stability, leading to high batch-to-batch variability and challenging the bench-to-bedside translation. Microfluidics has emerged as a powerful tool to produce PDNPs in a simple, reproducible, and cost-effective manner with excellent control over NP size. In this work, we designed and fabricated T- and Y-shaped Si-made microfluidics device and used it to produce pure NPs of three kinase inhibitors of different lipophilicity and water-solubility, namely imatinib, dasatinib and tofacitinib, without the use of colloidal stabilizers. PDNPs display sizes in the 90-350 nm range (dynamic light scattering) and a rounded shape (high-resolution scanning electron microscopy). Analysis by X-rays diffraction and differential scanning calorimetry confirmed that this method results in highly amorphous NPs. In addition, we show that the flow rate of solvent, the anti-solvent, and the channel geometry of the device play a key role in the size of the generated NPs.

Complete pathologic response (pCR) predicts the long-term outcome of neoadjuvant treated (NAC) breast cancer (BC) but is reached in <10% of hormone-receptor-positive patients. Biomarkers able to guide adjustment or interruption of an ineffective therapy are desired. Here, we evaluated whether shifts in the serum concentration of thymidine kinase 1 (sTK1) during NAC could be utilized as a biomarker. In the PROMIX trial, women with localized HER2- BC received neoadjuvant epirubicin/docetaxel in six cycles. sTK1 was measured with an ELISA in 54 patients at cycles 1-4 and in a total of 131 patients before and 48h after cycle 1. The prognostic significance of the results was evaluated by log-rank tests of Kaplan–Meier estimates. Treatment resulted in a 2-fold increase of sTK1 before and 3-fold increase 48h after the cycles, except for the first cycle, where half of patients reacted with a decrease (post/pre sTK1- ratio <1.12) and the other half reacted with an increase (ratio >1.12). OS rates in ER+ patients with ratios of >1.12 and <1.12 were 97.7% and 78% (p=0.005), respectively, and DFS rates were 90.7% and 68% (p=0.006), respectively. Thus, response of sTK1 at the first cycle of chemotherapy could be used both as an early biomarker for guidance of chemotherapy and for the study of inherent tumor chemo-sensitivity, which could predict long-term outcome prior to therapy.

Researchers have developed and used several three-dimensional (3D) culture systems, including spheroids, organoids, and tumoroids. Drug resistance is a crucial issue involving recurrence in cancer patients. Many studies on anticancer drugs have been done in 2D culture systems, where-as 3D cultured tumoroids have many advantages for assessing drug sensitivity and resistance. Here, we aim to investigate whether Cisplatin (a DNA crosslinker), Imatinib (a multiple tyro-sine kinase inhibitor), and 5-Fluorouracil (5-FU: an antimetabolite) alter tumoroid growth of metastatic colorectal cancer (mCRC). To establish a liquid-based 3D multiplexing reporter assay system, LuM1 (a murine mCRC cell line) was stably transfected with the Mmp9 promoter-driven ZsGreen reporter gene, which was designated as LuM1/m9 cells and cultured in NanoCulture Plate (NCP), a 3D culture device. The larger tumoroids were not sensitive to Cisplatin and ex-pressed ABCG2 (a marker of cancer stem cells, a.k.a. a drug efflux transporter), whereas smaller cell-aggregates were more sensitive to Cisplatin. Both Imatinib and Cisplatin significantly in-creased tumoroid growth (larger than 300 μm2) and Mmp9 promoter activity and were not cytotoxic to the mCRC tumoroids. On the other hand, 5-FU was cytotoxic to the tumoroids and significantly inhibited tumoroid growth, although not completely. Thus, platinum resistance and imatinib resistance in mCRC were modeled using the liquid-based 3D cultured tumoroid system. The tumoroid culture is useful and easily accessible for the assessment of drug sensitivity and resistance.

I have recently reiterated that the cross-bridge is a calcium ATPase that is inhibited by magnesium and this arises because in normal hearts Myosin binding Protein-C prevents the use of MgATP as rate limiting substrate ensuring that Ca²⁺ replaces Mg²⁺ in the excitation pathway. Here I revisit the studies on [Ca²⁺] dependency of ATPase and tension under diastolic stretch with a different conclusion on Hill coefficients. This reveals the underlying mechanisms of the Frank-Starling Law and Hypertrophic myopathy are not the same, the former being kinase controlled.

Choline kinase inhibitors are an important class of cytotoxic compounds useful for the treatment of different forms of cancer since aberrant choline metabolism is a feature of neoplastic cells. Here we present the characterization and the structure activity relationship of a series of non-symmetrical choline kinase inhibitors characterized by a 3-aminophenol moiety, bound to 4-(dimethylamino)- or 4-(pyrrolidin-1-yl)pyridinium cationic heads through several linkers. These derivatives were evaluated both for their inhibitory activity on the enzyme and for their antiproliferative activity in a panel of six human tumor cell lines. The compounds with the best inhibitory results were those connected to the linker by the N-atom (4a-h) and these results are supported by docking studies. The compounds with the best antiproliferative results were those connected to the linker by the O-atom (3a-h). On the other hand, as was predictable in both families, the inhibitory effect on the enzyme is greater the shorter the length of the linker, while in tumor cells, lipophilicity and choline uptake inhibition could play a decisive role. Interestingly compounds 3c and 4f, selected for both their ability to inhibit the enzyme and good antiproliferative activity, are endowed with a low toxicity in non-tumoral cells (e.g human peripheral lymphocytes) respect to cancer cells. These compounds were also able to induce to induce apoptosis in Jurkat leukemic cells without causing significative variations of cell cycle. It is worth to mention that these derivatives, beside their inhibitory effect on choline kinase, displayed a modest ability to inhibit choline uptake thus suggesting that this mechanism may also contribute to the observed cytotoxicity.

The New World (NW) mammarenavirus group includes several zoonotic highly pathogenic viruses, such as Junin (JUNV) or Machupo (MACV). Contrary to Old World mammarenavirus, these viruses are not able to completely suppress the innate immune response, and trigger a robust interferon (IFN)-I response via retinoic acid-inducible gene I (RIG-I). Nevertheless, pathogenic NW mammarenaviruses trigger a weaker IFN response than their non-pathogenic relatives do. RIG-I activation leads to upregulation of a plethora of IFN-stimulated genes (ISGs), which exert a characteristic antiviral effect either as lone effectors, or resulting from the combination with other ISGs or cellular factors. The dsRNA sensor-protein kinase receptor (PKR) is an ISG that plays a pivotal role in the control of the mammarenavirus infection. In addition to its well-known protein synthesis inhibition, PKR further modulates the overall IFN-I response against different viruses, including mammarenaviruses. For this study, we employed Tacaribe virus (TCRV), the closest relative of the human pathogenic JUNV. Our findings indicate that PKR does not only increase IFN-I expression against TCRV infection, but also affects the kinetic expression and the extent of induction of Mx1 and ISG15 at both levels, mRNA and protein expression. Moreover, TCRV fails to prevent the effect of PKR on viral protein translation and its viral titer is inhibited when PKR is pre-stimulated via IFN-I. Here, we provide first evidence of the specific immunomodulatory role of PKR over selected ISGs, altering the dynamic of the innate immune response course against TCRV. IMPORTANCE: The mechanisms for innate immune evasion are key for emergence and adaptation of human pathogenic arenaviruses, and highly pathogenic mammarenaviruses such as JUNV or MACV trigger a weaker IFN response than non-pathogenic mammarenaviruses. Within the innate immune response context, PKR plays an important role in sensing and restricting the infection of TCRV virus. Although the mechanism of PKR for protein synthesis inhibition is well described, its immunomodulatory role is less understood. In this study, we found that TCRV protein expression and viral propagation are inhibited from early times after infection, and when externally activated, PKR inhibits TCRV viral progeny production. Our present findings further characterize the innate immune response in absence of PKR, unveiling the role of PKR in defining the ISG profile after viral infection.

Crocodilepox virus (CRV) belongs to the Poxviridae family and mainly infects hatchling and juvenile Nile crocodiles. Most poxviruses encode inhibitors of the host antiviral protein kinase R (PKR), which is activated by viral double-stranded (ds) RNA formed during virus replication, resulting in the phosphorylation of eIF2 and subsequent shutdown of general mRNA translation. Because CRV lacks orthologs of known poxviral PKR inhibitors, we experimentally characterized one candidate (CRV157), which contains a predicted dsRNA-binding domain. Bioinformatic analyses indicated that CRV157 evolved independently from other poxvirus PKR inhibitors. CRV157 bound to dsRNA, co-localized with PKR in the cytosol, and inhibited PKR from various species. To analyze whether CRV157 could inhibit PKR in the context of a poxvirus infection, we constructed recombinant vaccinia virus strains that contain either CRV157 or a mutant CRV157 deficient in dsRNA binding in a strain that lacks PKR inhibitors. The presence of wild type CRV157 rescued vaccinia virus replication, while the CRV157 mutant did not. The ability of CRV157 to inhibit PKR correlated with virus replication and eIF2alpha phosphorylation. The independent evolution of CRV157 demonstrates that poxvirus PKR inhibitors evolved from a diverse set of ancestral genes in an example of convergent evolution.

Lung cancer is the most common malignancy across the world. The new era in lung cancer treatments, especially this past decade, has yielded novel categories of targeted therapy for specific mutations and adjuvant therapy, both of which have led to improved survival rates. In the present study, we review the changes and development of treatments, with a special focus on adjuvant therapy using tyrosine kinase inhibitors (TKIs) administered to non-small-cell lung carcinoma patients who had a complete resection of the tumor harboring a mutated epidermal growth factor receptor. The clinical trials are dating from the past (chemotherapy trials), present (TKIs) and future (ongoing trials).

Never in mitosis gene A-related kinase 2 (NEK2) a member of serine-threonine kinase protein mainly involved in the cell cycle process. Clinical studies revealed NEK2 overexpression in various tumour types, also NEK2 was reported for their association with genetic abnormalities like mitotic machinery deregulation and chromosomal instability. Besides NEK2 plays a key role in maintaining the transformed phenotype of cancer cells and chemo-resistance of several tumour types. Thus, NEK2 transcriptional profile is important for diagnosis, treatment, and prognosis stages of cancer studies. Screening of novel NEK2 inhibitor would be beneficial in developing the specific lead molecules. Our studies involved NEK2 transcriptional profile search, screening of druggable cavities in NEK2, Drug likeliness of mangrove derived naphthoquinone derivatives avicennoneA , avicennoneB , avicennoneC , avicennoneD , avicenoneE , avicennone F , and avicennone G , avicequinone A, stenocarpoquinone B , avicequinone C , avicenol A , avicenol C,brugine, apigenin, chrysin and molecular docking studies to assess MNC compounds binding efficacy towards NEK2. Mangrove derived compounds conferred the intermolecular hydrogen bond, Pi-alkyl,pi-cation interactions with NEK2 kinase domain region residues Tyr 19, Lys 37, Arg 164, Lys174. Nearly 200 kinase proteins contained this promising Cys 22 residue as its positioned in the catalytic site like NEK family proteins. Avicenna A, Avicennone G, Chrysin and Brugine formed the irreversible covalent binding with NEK2 through Cys 22, thus they can be considered as potential kinase inhibitors with the limited off-target response. But these MNC compounds need to be tested further in invitro and invivo studies to propose as potent NEK2 inhibitors.

Following the development of tyrosine kinase inhibitors (TKI), the survival of patients with chronic myeloid leukaemia (CML) drastically improved. With the introduction of these agents, CML is now considered a chronic disease, for some patients. Taking into consideration the side effects, toxicity, and high cost, discontinuing TKIs became a goal for patients with chronic phase CML. Patients who achieved deep molecular response (DMR) and discontinued TKI, remained in treatment-free remission (TFR). Currently, the data from the published literature demonstrate that 40-60% of patients achieve TFR, with relapses occurring within the first six months. In addition, almost all patients who relapsed regained a molecular response upon re-treatment, indicating TKI discontinuation is safe. However, there is still a gap in the understanding the mechanisms behind TFR, and whether there are prognostic factors that can predict the best candidates who qualify for TKI discontinuation with a view to keeping them in TFR. Furthermore, the information about a second TFR attempt and the role of gradual de-escalation of TKI before complete cessation is limited. This review highlights the factors predicting success or failure of TFR. In addition, it ex-amines the feasibility of a second TFR attempt after the failure of the first one, and the current guidelines concerning TFR in clinical practice.

Precise binding affinity predictions are essential for structure-based drug discovery (SBDD). Focal adhesion kinase (FAK) is a member of the tyrosine kinase protein family and is overexpressed in a variety of human malignancies. Inhibition of FAK using small molecules is a promising therapeutic option for several types of cancer. Here, we conducted computational modeling of FAK targeting inhibitors using 3-dimensional structure-activity relationship (3D-QSAR), molecular dynamics (MD), and hybrid topology-based free energy perturbation (FEP) methods. The structure-activity relationship (SAR) studies between the physicochemical descriptors and inhibitory activities of the chemical compounds were performed with reasonable statistical accuracy using CoMFA and CoMSIA. These are two well-known 3D-QSAR methods based on the principle of supervised machine learning (ML). Essential information regarding residue-specific binding interactions was determined using the MD and MM-PB/GBSA methods. Finally, physics-based relative binding free energy (〖∆∆G〗_RBFE^(A→B)) values of analogous ligands were estimated using the alchemical FEP simulation. An acceptable agreement was observed between the experimental and computed relative binding free energies. The overall results using ML and physics-based hybrid approaches could be useful for the rational optimization of accessible lead compounds with similar scaffolds targeting the FAK receptor.

The epigenetic modifications control the pathogenicity of human pathogenic fungi, which have been poorly studied in Mucorales; causative agents of mucormycosis. This order belongs to a group referred to as early-diverging fungi that are characterized by high levels of N6-methyldeoxyadenine (6mA) in their genome with dense 6mA clusters associated with actively expressed genes. AlkB enzymes can act as demethylases of 6mA in DNA, with the most remarkable eukaryotic examples being mammalian ALKBH1 and Caenorhabditis elegans NMAD-1. Mucor lusitanicus (formerly M. circinelloides f. lusitanicus) genome contains one gene, dmt1, and two genes, dmt2 and dmt3, encoding proteins homologs to C. elegans NMAD-1 and ALKBH1, respectively. The function of the three genes was analyzed by the generation of single and double deletion mutants for each gene. Multiple processes were studied in the mutants, but defects were only found in single and double deletion mutants for dmt1. In contrast to the wild-type strain, dmt1 mutants showed an increase of 6mA levels during the dimorphic transition, suggesting that 6mA regulates dimorphism in M. lusitanicus. Furthermore, the spores of dmt1 mutants challenged with macrophages underwent a reduction of polar growth, suggesting that 6mA also has a role during the spore-macrophage interaction that could be important in the infection process.

Aim To perform a comprehensive analysis of discordances between contrast-enhanced CT (ceCT) and 18F-FDG PET/CT in the evaluation of the extra-cerebral treatment monitoring in patients with stage IV melanoma. Materials and methods We conducted a retrospective monocentric observational study over a 3 years period in patients referred for 18F-FDG PET/CT and ceCT in the framework of therapy monitoring of immune checkpoint or tyrosine kinase inhibitors (ICIs or TKIs) as of January-2017. Imaging reports were analysed by two physicians in consensus. Anatomical site responsible for discordances, as well as induced changes in treatment were noted. Results Eighty patients were included and 195 couples of scans analysed. Overall, discordances occurred in 65 cases (33%). Eighty percent of the discordances (52/65) were due to 18F-FDG PET/CT scans upstaging the patient. Amongst these discordances, 17/52 (33%) led to change in patient’s management, the most frequent being radiotherapy of a progressing site. ceCT represented 13/65 (20%) of discordances and induced changes in patients’ management in 2/13 cases (15%). The more frequent anatomical site involved was subcutaneous for 18F-FDG PET/CT findings and lung or liver for ceCT. Conclusions Treatment monitoring with 18F-FDG

The involvement of the angiotensin II type 1 receptor in the Frank-Starling Law of the Heart, where the various activations are very limited, allows simple analysis of the kinase systems involved and thence extrapolation of the mechanism to that of angiotensin control of activation of cardiac and skeletal muscle contraction. The involvement of phosphorylation of the myosin light chain in the control of contraction is accepted but not fully understood. The involvement of troponin-I phosphorylation is also indicated but of unknown mechanism. There is no known signal for activation of myosin light chain kinase or Protein Kinase C-βII other than Ca²⁺/calmodulin but the former is constitutively active and thus has to be under control of a regulated inhibitor, the latter kinase may also be the same. Ca²⁺/calmodulin is not activated in Frank-Starling, i.e. there are no diastolic or systolic [Ca²⁺] changes. I suggest here that that the regulated inhibition is by myosin light chain phosphatase and/or β-arrestin. Angiotensin activation is by translocation of the β-arrestin from the sarcoplasm to the PM thus reducing its kinase inhibition action in the sarcoplasm, this reduced inhibition has been wrongly attributed to a mythical downstream agonist property of β-arrestin.

Phosphodiesterases (PDEs) hydrolyze cyclic nucleotides to modulate multiple signaling events in cells. PDEs are recognized to actively associate with cyclic nucleotide receptors (Protein Kinases, PK) in larger macromolecular assemblies referred to as signalosomes. Complexation of PDEs with PK generates an expanded active site which enhances PDE activity. This facilitates signalosome-associated PDEs to preferentially catalyze active hydrolysis of cyclic nucleotides bound to PK, and aid in signal termination. PDEs are important drug targets and current strategies for inhibitor discovery are based entirely on targeting conserved PDE catalytic domains. This often results in inhibitors with cross-reactivity amongst closely related PDEs and attendant unwanted side effects. Here, our approach targets PDE-PK complexes as they would occur in signalosomes, thereby offering greater specificity. Our developed fluorescence polarization assay has been adapted to identify inhibitors that block cyclic nucleotide pockets in PDE-PK complexes in one mode, and disrupt protein-protein interactions between PDEs and cyclic nucleotide activating protein kinases in a second mode. We tested this approach with three different systems: cAMP-specific PDE8-PKAR, cGMP-specific PDE5-PKG and dual-specificity RegA-RD complexes and ranked inhibitors according to their inhibition potency. Targeting PDE-PK complexes offers biochemical tools for describing the exquisite specificity of cyclic nucleotide signaling networks in cells.

Multiple sclerosis (MS) is a persistent inflammatory disease affecting the central nervous system. Bruton's tyrosine kinase (BTK) is an enzyme crucial in the communication process of B cells, which have a significant impact on the development of MS. Recently, BTK inhibitors have emerged as a promising treatment strategy for MS due to their ability to modulate the immune system. In this study, an extensive literature search was performed utilizing multiple databases, such as PubMed, Embase, and Scopus to examine the potential use of BTK inhibitors in managing cognitive disorders experienced by individuals with MS. We found that BTK inhibitors targeting microglia offer potential advantages for managing neurodegeneration associated with cognitive impairments in MS patients by slowing down degeneration processes effectively. Their ability to impact microglial function and penetrate the CNS positions them as promising candidates for improving cognitive function. This study suggests that BTK inhibitors provide new possibilities for managing inflammatory and neurodegenerative phases of MS through their effects on microglia activation pathways. Further research and clinical trials are necessary to fully explore their efficacy in treating MS while addressing barriers related to cognitive impairment.

We performed a systematic review and meta-analysis to evaluate the role of gastric acid suppressant use on outcomes of tyrosine kinase inhibitors (TKIs) and oral chemotherapy. We identified all researches evaluating the effect of GAS use on patients receiving oral chemotherapy or TKIs for solid tumors. The pooled hazard ratios (HRs) and 95% confidence interval (95% CI) for overall survival (OS) and progression-free survival (PFS) were calculated with fixed-effects or random-effects model. The study population included n=16 retrospective studies and 372,418 patients. Series concerned gastrointestinal tract tumors (n=5 studies), renal cell carcinomas (RCC, n=3 studies), non-small cell lung cancers (NSCLC, n=5 studies), and soft tissue sarcomas or mixed histologies solid tumors in n=3 studies. The pooled HRs for OS and PFS were 1.31 (95% CI: 1.20–1.43; P<01) and 1.3 (95%CI 1.07-1.57; P<0.01) for GAS and no GAS users, respectively. Only studies of EGFR mutated NSCLC patients receiving TKIs and those with colorectal cancer receiving oral chemotherapy showed a significant correlation between GAS and poor survival. Our study supports the evidence of a possible negative impact of concomitant GAS therapy on survival outcomes of patients receiving oral anti-cancer drugs.

The traditionally dismal outcome of acute myeloid leukemia (AML) patients carrying the FMS-related tyrosine kinase 3 (FLT3) mutations has been mitigated by the recent introduction into the clinics of tyrosine kinase inhibitors (TKI) such as midostaurin and gilteritinib. The present work summarizes the clinical data that led to the use of gilteritinib in clinical practice. Gilteritinib is a 2nd generation TKI with deeper single-agent activity than 1st generation drugs against both FLT3-ITD and TKD mutations, in human studies. Moreover, the phase I/II dose-escalation, dose-expansion Chrysalis trial showed an acceptable safety profile of gilteritinib (diarrhea, elevated aspartate aminotransferase, febrile neutropenia, anemia, thrombocytopenia, sepsis, and pneumonia) and a 49% overall response rate (ORR) in 191 FLT3-mutated relapsed/refractory (R/R) AML patients. In 2019, the pivotal ADMIRAL trial showed that the median overall survival was significantly longer in patients treated with gilteritinib than among those receiving chemotherapy (9.3 vs 5.6 months, respectively) and the ORR to gilteritinib was 67.6%, outperforming the 25.8% for chemotherapy arm and leading to the license for its clinical use by the US Food and Drug Administration. Since then, several real-world experiences confirmed the positive results in the R/R AML setting. Finally, gilterinib based combinations currently under investigation with several compounds (venetoclax, azacitidine, conventional chemotherapy, etc.) and some practical tips (maintenance after allogeneic transplantation, interaction with antifungal drugs, extramedullary disease, and onset of resistance) will be analyzed in detail in the review.

Vascular calcification (VC) is associated with increased cardiovascular risks in patients with chronic kidney disease (CKD). Sodium-glucose cotransporter 2 inhibitors, like empagliflozin, can improve cardiovascular and renal outcomes. We assessed the expression of Runt-related transcription factor 2 (Runx2), interleukin (IL)-1β, IL-6, AMP-activated protein kinase (AMPK), nuclear factor erythroid-2-related factor (Nrf2), and heme oxygenase 1 (HO-1) in inorganic phosphate–induced VC in mouse VSMCs to investigate the mechanisms underlying empagliflozin’s therapeutic effects. We evaluated biochemical parameters, mean artery pressure (MAP), pulse wave velocity (PWV), transcutaneous glomerular filtration rate (GFR), and histology in an in vivo mouse model with VC induced by an oral high-phosphorus diet following a 5/6 nephrectomy in ApoE−/− mice. Compared to the control group, empagliflozin-treated mice showed significant reductions in blood glucose, MAP, PWV, and calcification, as well as increased calcium and GFR levels. Empagliflozin inhibited osteogenic trans-differentiation by decreasing inflammatory cytokine expression and increasing AMPK, Nrf2, and HO-1 levels. Empagliflozin mitigates high phosphate–induced calcification in mouse VSMCs through the Nrf2/HO-1 anti-inflammatory pathway by activating AMPK. Animal experiments suggested that empagliflozin reduces VC in CKD ApoE−/− mice on a high-phosphate diet.

The majority of EGFR mutations (85–90%) are exon 19 deletions and L858R point mutation of exon 21, characterized by high sensitivity to EGFR-tyrosine kinase inhibitors (TKIs). Less is known about uncommon mutations (10-15% of EGFR mutations). Predominant mutation types in this category include exon 18 point mutations, exon 21 L861X, exon 20 insertions and exon 20 S768I. This group presents a heterogeneous prevalence, partly due to the different testing methods and to the presence of compound mutation, which in some cases leads to shorter overall survival and different sensitivity to different TKIs than simple mutations. EGFR-TKI sensitivity may also vary depending on the specific mutation and the tertiary structure of the protein. The best strategy remains uncertain and the data of TKIs efficacy are founded of few prospective and some retrospective series. Newer investigational agents are still under study and there are no other approved specific treatment targeting uncommon EGFR mutations. Defining the best treatment option for this patient population remain un unmet medical need. The objective of this review is to evaluate existing data on outcomes, epidemiology and clinical characteristics of lung cancer patients with rare EGFR mutations, with a focus on intracranial activity and response to immunotherapy.

Small molecule kinase inhibitors (SMKIs) are one of the heightened concerns in the field of drug research and development. There are seventy-nine (up to July 2023) small molecule kinase inhibitors have been approved by FDA and hundreds kinase inhibitors candidates in clinical trials, which shed light on the treatment of some major diseases. As an important strategy of drug design, computer-aided drug design (CADD) plays an indispensable role in the discovery of SMKIs. CADD methods, such as docking, molecular dynamic, pharmacophore have been applied to the design and optimization of small molecule kinase inhibitors. In the review, we provide an overview of recent advances in CADD and SMKIs; and the application of CADD for discovery of SMKIs.

Background: Newer personalized medicine including targeted therapies such as PARP inhibitors and CDK 4/6 inhibitors have shown to improve survival of breast and gynaecological cancer patients. However, efficacy outcomes may be hampered by treatment discontinuation due to targeted therapy-related adverse drug reactions or resistance. Studies suggest that add-on mistletoe (Viscum album L., VA) improves quality of life and ameliorates cytotoxic side effects of standard oncological therapy in cancer patients. The primary objective of this real-world data study was to determine the safety profile of targeted therapy in combination with add-on Helixor® VA therapy in breast and gynecological cancer patients. Methods: The present study is a real-world data study utilizing demographic and treatment data from the accredited national Network Oncology (NO) registry. The study has received ethics approval. The safety profile of targeted with or without Helixor® VA therapy as well as safety - associated variables were evaluated by univariate and adjusted multivariable regression analyses. Results: All stage breast or gynecological cancer patients (n = 242) were on average 54.5±14.2 years old. One hundred and sixty patients (66.1%) were in the control (CTRL, targeted therapy) and 82 patients (33.9%) in the combinational (COMB, targeted plus Helixor® VA therapy) group. The addition of Helixor® VA did not hamper the safety profile (χ2 = 0.107, p-value = 0.99) of targeted therapy. Furthermore, no adverse events and a trend towards an improved targeted therapy adherence were observed in the COMB group. Conclusions: The present study is the first of its kind showing the applicability of Helixor® VA in combination with targeted therapies. The results indicate that add-on Helixor® VA does not negatively alter the safety profile of targeted therapies in breast and gynaecological cancer patients.

Plasmodium species that cause malaria, a disease responsible for about half a million deaths per annum despite concerted efforts to combat it. The causative agent depends on type III beta phosphatidylinositol 4-kinase (PPI4K) during the development of merozoite. PPI4K is the only clinically validated Plasmodium kinase so far and its inhibitors are effective both in vitro and in vivo. In this work, a small library of ~22 000 fragments was virtually screened using PPI4K homology model to discover potential ligands of the enzyme. 16 virtual hits were selected based on ≤ -9.0 kcal/mol binding energy cut off and were subjected to similarity and substructure searching after they had passed PAINS screening. The derivatives obtained showed improved binding energies, which ranged from -10.00 to -13.80 kcal/mol. Moreover, the topmost ranking compound 31, with interesting drug-like quality was stable within the protein’s binding cavity during the 10 ns molecular dynamics simulation period. In addition, analysis of its binding pose revealed some unique binding interactions with PPI4K active site residues as the basis for the observed improved binding affinity. Overall, compound 31 appears to be a viable starting point for the development of PPI4K inhibitors with antimalarial activity.

Cholesterol biosynthesis inhibitors (statins) protect hypercholesterolemic patients from developing active tuberculosis, suggesting that these drugs could help the host to control the pathogen at the initial stages of the disease. This work studies the effect of fluvastatin on the early response of healthy peripheral blood mononuclear cells (PBMC) to inactivated Mycobacterium tuberculosis (Mtb) H37Ra. We found that in fluvastatin-treated PBMC, most monocytes/macrophages became foamy cells that overproduce NLRP3 inflammasome components in the absence of immune stimulation, evidencing important cholesterol metabolism/immunity connections. When both fluvastatin-treated and untreated PBMC were exposed to Mtb, a subset of macrophages quickly captured large amounts of bacilli and died, concentrating the bacteria in necrotic areas. In fluvastatin-untreated cultures, most of the remaining macrophages became epithelioid cells that isolated these areas of cell death in granulomatous structures that barely produced IFN-gamma. By contrast, in fluvastatin-treated cultures, foamy macrophages surrounded the accumulated bacteria to degrade them, markedly activated caspase-1 and elicited a potent IFNgamma/cytotoxic response. In rabb-gammaits immunized with the same bacteria, fluvastatin therapy increased the tuberculin test response. We conclude that statins enhance macrophage effectiveness to control Mtb supported by adaptive immunity, offering a promising tool in the design of alternative therapies to fight tuberculosis.

mTORC1 regulates mammalian cell metabolism and growth, in response to diverse environmental stimuli. Nutrient signals control the localization of mTORC1 onto lysosome-surface scaffolds that are critically implicated in its amino acid-dependent activation. Arginine, Leucine and S-Adenosyl-Methionine (SAM) can serve as major mTORC1-signaling activators, with SAM binding to SAMTOR (SAM + TOR), a fundamental SAM sensor, abolishing protein’s (SAMTOR’s) inhibitory action(s) against mTORC1, thereby releasing its (mTORC1) kinase activity. Given the lack of knowledge regarding the role of SAMTOR in invertebrates, we have, in silico, identified the Drosophila SAMTOR homologue (dSAMTOR) and have, herein, genetically targeted it, through utilization of the GAL4/UAS transgenic tool. Survival profiles and negative geotaxis patterns were examined both in control and dSAMTOR-downregulated adult flies, during aging. One of the two gene-targeted schemes resulted in lethal phenotypes, whereas the other one caused rather moderate pathologies in most tissues. Screening of head-specific kinase activities, via PamGene technology application, unveiled the significant upregulation of several kinases, including the dTORC1 characteristic substrate, dp70S6K, in dSAMTOR-downregulated flies, thus strongly supporting the inhibitory dSAMTOR function(s) upon dTORC1/dp70S6K signaling axis in Drosophila-brain settings. Importantly, genetic targeting of the Drosophila BHMT -bioinformatics- counterpart (dBHMT), an enzyme that catabolizes Betaine to produce Methionine (the SAM precursor), led to severe compromises in fly longevities, with glia-, motor neuron- and muscle-specific dBHMT downregulations exhibiting the strongest effects. Abnormalities in wing-vein architectures were also detected in dBHMT-targeted flies, thereby justifying their notably reduced negative geotaxis capacities, herein, observed, mainly in the brain-(mid)gut axis. In vivo adult-fly exposure to clinically relevant doses of Methionine revealed the mechanistic synergism of decreased dSAMTOR and increased Methionine levels in pathogenic longevity, thus rendering (d)SAMTOR an important component in Methionine-associated disorders, including Homocystinuria(s).

Aim To perform a comprehensive analysis of discordances between contrast-enhanced CT (ceCT) and 18F-FDG PET/CT in the evaluation of the extra-cerebral treatment monitoring in patients with stage IV melanoma. Materials and methods We conducted a retrospective monocentric observational study over a 3 years period in patients referred for 18F-FDG PET/CT and ceCT in the framework of therapy monitoring of immune checkpoint or tyrosine kinase inhibitors (ICIs or TKIs) as of January-2017. Imaging reports were analysed by two physicians in consensus. Anatomical site responsible for discordances, as well as induced changes in treatment were noted. Results Eighty patients were included and 195 couples of scans analysed. Overall, discordances occurred in 65 cases (33%). Eighty percent of the discordances (52/65) were due to 18F-FDG PET/CT scans upstaging the patient. Amongst these discordances, 17/52 (33%) led to change in patient’s management, the most frequent being radiotherapy of a progressing site. ceCT represented 13/65 (20%) of discordances and induced changes in patients’ management in 2/13 cases (15%). The more frequent anatomical site involved was subcutaneous for 18F-FDG PET/CT findings and lung or liver for ceCT. Conclusions Treatment monitoring with 18F-FDG

Antimicrobial application of carbon nanomaterials such as carbon nanotubes (CNTs), capped CNT, C60 fullerene, and C70 fullerene are increasing owing to their low cytotoxicity properties compared to other nanomaterials such as metallic nanoparticles. Enhanced mechanical property and antibacterial activity can be caused by incorporation of CNTs in 3-dimentional (3D) printed nanocomposites (NCs). Interruption of bacterial membrane resulting from cylindrical shape and high aspect ratio properties has been found as the prominent antibacterial mechanism of CNTs. However, unraveling interaction of CNT, capped CNT, C60 fullerene, and C70 fullerene with virulence factors of the main bacterial pathogenesis has not yet been known. Therefore, in the present study, interactions of CNT, capped CNT, C60, and C70 with the eight virulence factors including protein kinase A and ESX-secreted protein B of Mycobacterium tuberculosis, pseudomonas elastase and exotoxin A of Pseudomonas aeruginosa, alpha-hemolysin and penicillin binding protein 2a of Staphylococcus aureus and shiga toxin 2a and heat-labile enterotoxin of Escherichia coli have been evaluated by molecular docking method of AutoDock Vina. This study disclosed the binding strength followed the sequence CNT &gt; capped CNT &gt; C70 &gt; C60 towards alpha-hemolysin of S. aureus compared to other virulence factors with values of -19.6, -18.8, -13.6, and -12.8 kcal/mol, respectively. The lowest and highest binding affinity were found for CNT against 1TII and 1MWT by binding energy values of 97.4 and -20.1 kcal/mol. The stability of CNT-1MWT complex at the different times has resulted according to the normal mode analysis of elNémo and iMOD servers. Future studies should be focused on improving cellular uptake of CNTs, capped CNTs, C70, and C60 by surface functionalization for active targeting of bacteria.

Calcium/calmodulin (CaM)-dependent protein kinase kinase 2 (CaMKK2) regulates bone remodeling through its effects on osteoblasts and osteoclasts. However, its role in osteocytes, the most abundant bone cell type, and the master regulator of bone remodeling, remains unknown. Here we report that the conditional deletion of CaMKK2 from osteocytes using Dentine matrix protein 1 (Dmp1)-8kb-Cre mice led to enhanced bone mass only in female mice owing to a suppression of osteoclasts. Conditioned media isolated from female CaMKK2-deficient osteocytes inhibited osteoclast formation and function in in vitro assays, indicating a role for osteocyte-secreted factors. Proteomics analysis revealed significantly higher levels of extracellular calpastatin, a specific inhibitor of calcium-dependent cysteine proteases calpains, in female CaMKK2 null osteocyte conditioned media, compared to media from female control osteocytes. Further, exogenously added non-cell permeable recombinant calpastatin domain I elicited a marked, dose-dependent inhibition of female wild-type osteoclasts and depletion of calpastatin from female CaMKK2-deficient osteocyte conditioned media reversed the inhibition of matrix resorption by osteoclasts. Our findings reveal a novel role for extracellular calpastatin in regulating female osteoclast function and unravel a novel CaMKK2-mediated paracrine mechanism of osteoclast regulation by female osteocytes.

The emergence of chemoresistance in neoplastic cells is one of the major obstacles in cancer therapy. Autophagy was recently reported as one of the mechanisms that promote chemoresistance in cancer cells by protecting from apoptosis and driving senescence. Thus, understanding the role of autophagy and its underlying signaling pathways is crucial for the development of new therapeutic strategies to overcome chemoresistance. We have previously reported that PKCη is a stress-induced kinase that confers resistance in breast cancer cells against chemotherapy by inducing senescence. Here we show that PKCη promotes autophagy induced by ER and oxidative stress and facilitates the transition from autophagy to senescence. We demonstrate that PKCη knockdown reduces both the autophagic flux and markers of senescence. Additionally, using autophagy inhibitors, such as chloroquine and 3-methyladenine, we show that PKCη and autophagy are required for establishing senescence in MCF-7 in response to oxidative stress. Different drugs used in the clinic are known to induce autophagy and senescence in breast cancer cells. Our study proposes PKCη as a target for therapeutic intervention, acting in synergy with autophagy-inducing drugs, to overcome resistance and enhance cell death in breast cancer.

Metastasis is the main cause of anti-cancer therapies failure, leading to unfavorable prognosis for patients. The true challenge to increase cancer patient life expectancy by making cancer a chronic disease with periodic but manageable relapses, relies on the development of efficient therapeutic strategies specifically directed against key targets in the metastatic process. Traditional chemotherapy with classical alkylating agents, microtubule inhibitors and antimetabolites has demonstrated its limited efficacy against metastatic cells due to their capacity to select chemo-resistant cell populations that undergo epithelial to mesenchymal transition (ETM) thus promoting the colonization of distant sites that in turn sustain the initial metastatic process. This scenario has prompted efforts aimed at discovering a wide variety of small molecules as potential anti-metastatic drugs directed against more specific targets known to be involved in the various stages of metastasis. In this short reviews we will give an overview of the most recent advances related to three main large families of antimetastatic small molecules: intracellular tyrosine kinase inhibitors, integrin antagonists and small interfering (siRNAs). Although the majority of these small molecules are not yet approved and not available in the drug market, any information related to their stage of development could represent a precious and valuable tool to identify new targets in the endless fight against metastasis.

Citation: Lastname, F.; Lastname, F.; Last-name, F. Title. Phar-maceuticals 2022, 15, x. https://doi.org/10.3390/xxxxx Academic Editor: Firstname Lastname Received: date Accepted: date Published: date Publisher’s Note: MDPI stays neutral with regard to jurisdic-tional claims in pub-lished maps and insti-tutional affiliations. Copyright: © 2023 by the authors. Submitted for possible open access publication under the terms and conditions of the Creative Commons 1Olga Gumkowska-Sroka, 2Kacper Kotyla, 3Ewa Mojs, 2Klaudia Palka, 1,2*Przemysław Kotyla 1. Department of Rheumatology and Clinical Immunology Voivodeship Hospital No5 in Sosnowiec Medical University of Silesia Katowice, Poland 2. Department of Internal Medicine Rheumatology and Clinical Immunology Medical University of Silesia Katowice , Poland 3. 3. Department of Clinical Psychology, Poznan University of Medical Sciences Poznan, Poland *Correspondence: Prof Przemysław Kotyla Department of Internal Medicine Rheumatology and Clinical Immunology Medical University of Silesia, Katowice, Poland- Summary Systemic sclerosis is a connective tissue disease of unknown origin and unpredictable course, with both cutaneous and internal organ manifestations. In spite of enormous progress in rheumatology and clinical immunology, the background of diseases is largely unknown and no specific therapy exists. The therapeutic approach to the disease is aimed to treat and preserve the function of internal organs, and this approach is commonly referred to as an organ-based treatment. However, in modern times data from the other branches of medicine may help to treat disease-related complications, making it possible to find a group of drugs to be utilized in the treatment of the disease. In this review, we present possible therapeutic options aiming to stop the progression of fibrotic processes, restore aberrant immune response, stop improper signalling from proinflammatory cytokines, and halt the production of disease-related autoantibodies.

(1) Background: Heart failure (HF) is the final stage of multiple cardiac diseases, which has now become a severe public health problem worldwide. β-Adrenergic receptor (β-AR) overactivation is a major pathological factor associated with multiple cardiac diseases and mediates cardiac fibrosis and inflammation. Previous study has demonstrated that Bruton's tyrosine kinase (BTK) mediated cardiac fibrosis by TGF-β related signal pathways, indicating that BTK was a potential drug target for cardiac fibrosis. Zanubrutinib, a second-generation BTK inhibitor, has shown anti-fibrosis effects in previous research. However, it is unclear whether Zanubrutinib can alleviate cardiac fibrosis induced by β-AR overactivation;(2) Methods: In vivo: Male C57BL/6J mice were treated with or without the β-AR agonist isoproterenol (ISO) to establish cardiac fibrosis animal model. Here, results showed that BTK inhibitor Zanubrutinib (ZB) had a great effect on cardiac fibrosis and inflammation induced by β-AR. In vitro: Results showed that ZB alleviated β-AR-induced cardiac fibroblast activation and macrophage proinflammatory cytokine production. Further mechanism studies demonstrated that ZB inhibited β-AR-induced cardiac fibrosis and inflammation by BTK、STAT3、NF-κB and PI3K/Akt signal pathways both in vivo and in vitro; (4) Conclusions: our research provides evidence that ZB ameliorates β-AR-induced cardiac fibrosis and inflammation.

Alcohol effects on hepatic lipid metabolism through various mechanisms, leading synergistically to an accumulation of fatty acids (FA) and triglycerides. Obesity, as well as, the dietary fat [saturated fatty acids (FA) versus poly-unsaturated fatty acids (PUFA)] may modulate the hepatic fat. Alcohol inhibits adenosine monophosphate activated kinase (AMPK). AMPK activates peroxisome proliferators activated receptor a (PPARα) and leads to a decreased activation of sterol regulatory element binding protein 1c (SRABP1c). The inhibition of AMPK, and thus of PPARα results in an inhibition of FAs oxidation. This ß-oxidation is further reduced due to mitochondrial damage induced through cytochrome P4502E1 (CYP2E1) driven oxidative stress. Furthermore, the synthesis of FAs is stimulated through an activation of SHREP1. In addition, alcohol consumption leads to a reduced production of adiponectin in adipocytes due to oxidative stress and to an increased mobilization of FAs from adipose tissue and from the gut as chylomicrons. On the other side, the secretion of FAs via very low density lipoproteins (VLDL) from the liver is inhibited by alcohol. Alcohol also affects signal pathways such as early growth response 1 (Egr-1) associated with the expression of tumour necrosis factor  (TNF), and the mammalian target of rapamycin (mTOR) a key regulator of autophagy. Both have influence the pathogenesis of alcoholic fatty liver. Alcohol-induced gut dysbiosis is contributing to the severity of ALD by increasing metabolism of ethanol in the gut and promoting intestinal dysfunction. Moreover, pathogen associated molecular paterns (PAMPS) via specific Toll-like receptors (TLR) bacterial overgrowth is leading to the translocation of bacteria. Endotoxins, and toxic ethanol metabolites enter the enterohepatic circulation reaching the liver, inducing the activation of the nuclear factor kappa-B (NFB) pathway. Pro-inflammatory cytokines released in the process contribute to inflammation and fibrosis. In addition, cellular apoptosis is inhibited in the favor of necrosis.

Pediatric Autoimmune Neuropsychiatric Disorders Associated with Streptococcal Infections (PANDAS) is one of the most controversial diseases in pediatric rheumatology. Despite its first description being published more than 25 years ago as the sudden and rapid onset of obsessive-compulsive disorder (OCD) and/or tic disorder symptoms as complications of a Group A beta-hemolytic Streptococcus (GAS) infection, precise epidemiological data are still lacking, and there are no strong recommendations for its treatment. Recent advances in the comprehension of PANDAS pathophysiology are largely attributable to animal model studies and the understanding of the roles of Ca++/calmodulin-dependent protein kinase (CaM kinase) II, disrupted dopamine re-lease in the basal ganglia, and striatal cholinergic interneurons. The diagnosis of PANDAS should be made after an exclusion process and should include prepubescent children with a sudden onset of OCD and/or a tic disorder, with a relapsing/remitting disease course, a clear temporal association between GAS infection and onset or exacerbation of symptoms, and the association with other neurological abnormalities such as motoric hyperactivity and choreiform movements. Antibiotic medications are the primary therapeutic modality. Nonetheless, there is a paucity of randomized studies and validated data, resulting in a scarcity of solid recommendations.

Methicillin-resistant Staphylococcus aureus (MRSA) exerts a considerable concern in healthcare and the community. Globally MRSA strains cause numerous infections with high morbidity and mortality rates. The success of MRSA is a result of the extensive production of virulence factors. With the limited treatment options for the MRSA infection and growing resistance problem, an urgent call is needed to search for an alternative to antibiotics. One of the promising strategies is targeting the quorum sensing (QS) system, of the MRSA, by using a dendrimer loaded with kinase inhibitor peptide. This study formulates a poly-amidoamine dendrimers (PAMAM) G4 dendrimers loaded with Quorum Quencher (QQ), QQ3 peptide which is a Histidine Kinase inhibitor. The physicochemical properties of the formula were characterized using different in-vitro analysis method such as Zetasizer and transmission electron microscope (TEM). The antibacterial activity and anti-virulence effect of the formula was tested against MRSA and different mutant stains using growth assay, haemolysis assay and biofilm assay. Overall, QQ3-G4 complex showed antibacterial and anti-virulence activity and it can be a potential alternative treatment to overcome antimicrobial resistance and treat MRSA infection.

The initiation reactions of DNA synthesis are central processes during human chromosomal DNA replication. They are separated into two main processes: the initiation events at replication origins, the start of the leading strand synthesis for each replicon, and the numerous initiation events taking place during lagging strand DNA synthesis. In addition, a third mechanism is the re-initiation of DNA synthesis after replication fork stalling, which takes place when DNA lesions hinder the progression of DNA synthesis. The initiation of leading strand synthesis at replication origin is regulated at multiple levels, from the origin recognition to the assembly and activation of replicative helicase, the Cdc45-MCM2-7-GINS (CMG) complex. In addition, the multiple interactions of the CMG complex with the eukaryotic replicative DNA polymerases such as DNA polymerase α-primase (Pol α), DNA polymerase δ and ε at replication forks play pivotal roles in the mechanism of initiation reactions including the initiation of signalling unperturbed and stalled replication forks, ‘replication stress’ events, via ATR (ATM-Rad 3-related protein kinase). All these processes are essential for the accurate transfer of the cells’ genetic information to their daughters. Failures and dysfunctions in these processes gives rise to genome instability followed by genetic diseases including cancer. Thus, in their review ‘Hallmarks of Cancer: New Dimensions‘ Hanahan and Weinberg (2022) called genome instability a fundamental function in the development process of cancer cells. In recent years, the understanding of the initiation processes and mechanisms of human DNA replication has made substantial progress at all levels which will be discussed in the review.

Rarely has a chemical elicited as much controversy as has dichloroacetate (DCA). DCA was initially considered a dangerous toxic industrial waste product. Then it was a potential treatment for lactic acidosis. However, the main controversies started in 2008 when DCA was found to have anti-cancer effects in experimental animals. These publications showed contradictory results in vivo and in vitro, so that a thorough consideration of this compound in cancer is merited. Despite 50 years of experimentation, DCA’s future in therapeutics is uncertain. Without adequate clinical trials and lack of health authorities’ approval, DCA has been introduced in off -label cancer treatments in alternative medicine clinics in Canada, Germany, and other European countries. The lack of well-planned clinical trials and its use by people without medical training has discouraged consideration by the scientific community. There are limited thorough clinical studies of DCA, and many publications are individual case reports. Case reports of DCA benefits against cancer have been increasing recently. Furthermore, it has been shown that DCA synergizes with conventional treatments and other repurposable drugs. Beyond the classic DCA target, pyruvate dehydrogenase kinase, new target molecules have also been recently discovered. These findings have renewed interest in DCA. This paper explores whether existing evidence justifies further research on DCA for cancer treatment and it explores the role DCA may play in oncology treatment.

Casein Kinase ІІ (CK2) is one of the most versatile kinases. Its involvement in almost all cellular pathways makes it the Master Regulator of biochemical processes in a cell. This kinase is essential in regulating inflammation, cell differentiation, and cell cycle regulation. Often simultaneously. Its emerging role in senescence also indicates its function's centrality in cellular metabolism. The strategy to target this kinase to treat musculoskeletal disorders seems effective. These disorders often include a component of inflammation, dysregulated cell differentiation, and aging. This review focuses on CK2 target discovery and the diversity of its substrate interactions. We then transition toward the implication of CK2 in musculoskeletal disorders. Through a summary of current strategies for CK2 targeting with a new approach, we discuss the potential aspects that could be addressed in future drug research.

PTK6, a Non-Receptor–Tyrosine-Kinase, modulates the pathogenesis of breast and prostate cancers, and is recognized as a biomarker of breast cancer prognosis. There are over 30 known substrates of PTK6, including signal transducers, transcription factors and RNA binding proteins. Many of these substrates are known drivers of other cancer types such as colorectal cancer. Colon and rectal tumours also express higher levels of PTK6 than the normal intestine suggesting a potential role in tumorigenesis. However, the importance of PTK6 in colorectal cancer remains unclear. PTK6 inhibitors such as XMU-MP-2 and Tilfrinib have demonstrated potency and selectivity in breast cancer cells when used in combination with chemotherapy, indicating the potential for PTK6 targeted therapy in cancer. However, most of these inhibitors are yet to be tested in other cancer types. Here, we discuss the current understanding of the function of PTK6 in normal intestinal cells compared with colorectal cancer cells. We review existing PTK6 targeting therapeutics and explore the possibility of PTK6 inhibitory therapy for colorectal cancer.

Avenanthramides (Avns) and its derivatives, a group of polyphenolic compounds found abundantly in Oats (Avena sativa Linn.) have emerged as a promising candidate for neuroprotection due to their immense anti-oxidant, anti-inflammatory, and anti-apoptotic properties. Neurodegenerative diseases (NDDs), characterized by progressive degeneration of neurons, present a significant global health burden with limited therapeutic options. The phosphoinositide 3-kinase (PI3K) signalling pathway plays a crucial role in cell survival, growth, and metabolism, making it an attractive target for therapeutic intervention. Dysregulation of PI3K signalling has been implicated in the pathogenesis of various NDDs including Alzheimer's and Parkinson's disease. Avns has been shown to modulate PI3K/AKT signalling, leading to increased neuronal survival, reduced oxidative stress, and improved cognitive function. This review explores the potential of Avns polyphenols, as modulators of the PI3K signalling pathway focusing on their beneficial effects against NDDs. Further, we outlined the need for clinical exploration to elucidate the specific mechanisms of Avns action on the PI3K/AKT pathway and its potential interactions with other signalling cascades involved in neurodegeneration. Based on the available literature using relevant keywords from Google Scholar, PubMed, Scopus, Science Direct, and Web of Science, our review emphasizes the potential of Avns as a therapeutic strategy for NDDs and warrants further investigation and clinical exploration.

A number of agents, including immune checkpoint inhibitors, have become available for the treatment of hepatocellular carcinoma (HCC). However, the objective response rate of these drugs is currently only 30% to 40%, with a high incidence of side effects. There are also no prac-tical biomarkers to predict their therapeutic effects. Most of the systemic therapies for HCC are performed in general hospitals without research facilities. Such hospitals can perform imaging tests, like CT and MRI, as well as pathological diagnosis using tumor tissue sampling and im-munohistochemical staining. However, analyzing tumor genomic or transcriptomic profiles is difficult because of limitations in facilities, personnel, and cost. Therefore, in this review, we provide an overview of the wide range of research that has been conducted on HCC biomarkers from blood, tissue, or imaging information that can be used practically in general hospitals for predicting the therapeutic effect of systemic therapies before treatment begins. For general hos-pitals that treat HCC patients, we recommend conducting treatment after assessing the state within the tumor tissue as much as possible by collecting blood and tissue samples and per-forming pre- treatment MRI image evaluations.

Rabson-Mendenhall syndrome is a rare autosomal recessive disorder characterized by severe insulin resistance, resulting in early-onset diabetes mellitus. We are reporting the first case of Rabson-Mendenhall syndrome in a Paraguayan patient. The patient is a 5-year-old girl who presented with hypertrichosis, acanthosis nigricans, and nephrocalcinosis. Genetic testing by NGS revealed two pathogenic variants in exons 2 and 18 of the INSR gene; c.332G&gt;T (p. Gly111Val) and c.3485C&gt;T (p. Ala1162Val), in combined heterozygosis. The novel INSR c. 332G&gt;T variant leads to the substitution of glycine to valine at position 111 in the protein, and multiple in silico software predicted it as pathogenic. The c. 3485C&gt;T variant leads to the substitution of alanine to valine at position 1162 in the protein previously described for insulin resistance and Rabson-Mendenhall syndrome. The management of Rabson-Mendenhall syndrome is particularly challenging in children, and the use of metformin is often limited by its side effects. The patient was managed with nutritional measures due to the early age of onset. This report expands the knowledge of Rabson-Mendenhall syndrome to the Paraguayan population and adds a novel pathogenic variant to the existing literature.

The dual leucine zipper kinase (DLK) alias mitogen-activated protein 3 kinase 12 (MAP3K12) has gained much attention in recent years. DLK belongs to the mixed lineage kinases, characterized by homology to serine/threonine and tyrosine kinase but exerts serine/threonine kinase activity. DLK has been implicated in many diseases including several neurodegenerative diseases, glaucoma, and diabetes mellitus. As a MAP3K, it is generally assumed that DLK becomes phosphorylated and activated by upstream signals and phosphorylates and activates itself, the downstream serine/threonine MAP2K, and ultimately MAPK. In addition, other mechanisms such as protein-protein interactions, proteasomal degradation, dephosphorylation by various phosphatases, palmitoylation, and subcellular localization have been shown to be involved in the regulation of DLK activity or its fine-tuning. In the present review, the diverse mechanisms regulating DLK activity will be summarized to provide a better insight into DLK action and possibly new targets to modulate DLK function.

Polo-like kinase 1 (PLK1) plays a pivotal role in cell division regulation and emerges as a promising therapeutic target for cancer treatment. Consequently, the development of small-molecule inhibitors targeting PLK1 has become a focal point in contemporary research. The adenosine triphosphate (ATP)-binding site and the polo-box domain in PLK1 present crucial interaction sites for these inhibitors, aiming to disrupt the protein's function. However, designing potent and selective small-molecule inhibitors can be challenging, requiring a deep understanding of protein-ligand interaction mechanisms at these binding sites. In this context, our study leverages the fragment molecular orbital (FMO) method to explore these site-specific interactions in-depth. Through the FMO approach, we used the FMO method to elucidate the molecular mechanisms of small-molecule drug binding to these sites to design PLK1 inhibitors that are both potent and selective. Our investigation further entailed a comparative analysis of various PLK1 inhibitors, each characterized by distinct structural attributes. This comparison enhanced our understanding of the structure-activity relationships of these inhibitors, underscoring the efficacy of the FMO method in identifying critical binding features and predicting binding modes for small-molecule ligands. Furthermore, Our research spotlighted "hot spot" residues instrumental for selective and robust PLK1 binding. Our findings offer profound insights, priming the rational design of innovative potential PLK1 inhibitors with significant implications for developing anticancer therapeutics.

Eosinophils in peripheral blood account for 0.3-5% of leukocytes, which is equivalent to 0.05-0.5 x 109/l. A count equal or above 0.5 x 109/l is considered eosinophilia, while a count equal or above 1.5 x 109/l is defined as hypereosinophilia. In bone marrow, eosinophilia is considered when eosinophils make up more than 6% of the total nuclear cells. In daily clinical practice, the most common causes of reactive eosinophilia are non- hematologic, whether non-neoplastic (allergic diseases, drugs, infections or immunological diseases) or neoplastic (solid tumors). Eosinophilia associated with a haematological malignancy may be reactive or secondary to the production of eosinophilopoietic cytokines, and this is mainly seen in lymphoid neoplasms (Hodgkin lymphoma, mature T-cell neoplasms, lymphoid variant of hypereosinophilic syndrome and B-acute lymphoblastic leukemia/lymphoma). Eosinophilia associated with a haematological malignancy may also be neoplastic or primary, derived from the malignant clone, usually in myeloid neoplasms or with origin in stem cell (myeloid/lymphoid neoplasms with eosinophilia and tyrosine kinase gene fusions, acute myeloid leukemia with Core Binding Factor translocations, mastocytosis, myeloproliferative neoplasms, myelodysplastic/myeloproliferative neoplasms and myelodysplastic neoplasms). There are no concrete data in standardized cytological and cytometric procedures that could predict whether eosinophilia is reactive o clonal. The verification is usually indirect, based on the categorization of the accompanying hematologic malignancy. This review focuses on the broad differential diagnosis of haematological malignancies with eosinophilia.

The term neurodegeneration with brain iron accumulation (NBIA) brings together a broad set of progressive and disabling neurological genetic disorders in which iron is deposited preferentially in certain areas of the brain. Among NBIA disorders, the most frequent subtype is pantothenate kinase-associated neurodegeneration (PKAN) caused by pathologic variants in the PANK2 gene codifying the enzyme pantothenate kinase 2 (PANK2). Nowadays, there are no effective treatments to stop the progression of these diseases. This review discusses the utility of patient-derived cell models as a valuable tool for the identification of commercial pharmacological or natural compounds for implementing polytarget precision medicine in PKAN. In the last years, several studies have described that PKAN patient-derived fibroblasts manifest the main pathological changes associated with the disease including intracellular iron overload. Interestingly, treatment of mutant cell cultures with various supplements such as pantothenate, pantethine, thiamine, L-carnitine, vitamin E, omega 3, and -lipoic acid improved all pathophysiological alterations in PKAN fibroblasts with residual expression of the PANK2 enzyme. The information provided by pharmacological screenings in patient-derived cellular models can help to optimize therapeutic strategies in individual PKAN patients.

Purpose: Inducible Slc4a11 KO leads to corneal edema by disruption of the pump and barrier functions of the corneal endothelium (CE). The loss of Slc4a11 NH3-activated mitochondrial uncoupling leads to mitochondrial membrane potential hyperpolarization-induced oxidative stress. The goal of this study is to investigate the link between oxidative stress and failure of pump and barrier functions and test different approaches to revert the process. Methods: Mice homozygous for Slc4a11 Flox and Estrogen receptor –Cre Recombinase fusion protein alleles at 8 weeks of age were fed Tamoxifen (Tm) enriched chow (0.4 g/Kg) for 2 weeks, and controls were fed normal chow. During the initial 14 days, Slc4a11 expression, corneal thickness (CT), stromal [lactate], Na+-K+ ATPase activity, mitochondrial superoxide levels, expression of lactate transporters, and activity of key kinases were assessed. In addition, barrier function was assessed by fluorescein permeability, ZO-1 tight junction integrity, and cortical cytoskeleton F-actin morphology. Results: Tm induced a rapid decay in Slc4a11 expression that was 84% complete at 7 days and 96% at 14 days of treatment. Superoxide levels increased significantly by day 7; CT and fluorescein permeability by day 14. Tight junction ZO-1 distribution and cortical cytoskeleton were disrupted at day 14 concomitant with decreased expression of Cldn1 yet an increase in tyrosine phosphorylation. Stromal lactate increased by 60%, Na+-K+ATPase activity decreased by 40%, and expression of lactate transporters MCT2 and MCT4 significantly decreased, but MCT1 was unchanged at 14 days. Src kinase was activated but not Rock, PKCα, JNK, or P38Mapk. Mitochondrial antioxidant Visomitin (SkQ1, mitochondrial targeted antioxidant) or Src kinase inhibitor eCF506 significantly slowed the increase in CT, with concomitant decreased stromal lactate retention, improved barrier function, reduced Src activation and Cldn1 phosphorylation, and rescued MCT2 and MCT4 expression. Conclusions: Slc4a11 KO-induced CE oxidative stress triggered increased Src kinase activity that results in perturbation of pump components and barrier function of the CE.

Cardiotonic steroids (CTS) are a group of steroid compounds derived from certain plants and animals. CTS are selective inhibitors of Na,K-ATPase, so for a long time they were widely used for medical purposes to treat heart failure and some other diseases. However, over time, doctors gradually began to refuse this group of drugs due to their narrow therapeutic range and a number of serious side effects. However, in parallel with the rise and fall of interest in CTS as a drug, interest in them as a single class of high-affinity Na,K-ATPase inhibitors has only increased. Numerous data on the effect of CTS on signaling cascades and cell viability made impelling a search for a rationale for the existence of such a subtle biological regulator. A large number of studies devoted to the potential use of CTS in the treatment of neurodegenerative, oncological, immune and other diseases "revitalized" the already "written off" group of the drugs. If initially the role of exogenous (mainly plant origin) CTS was studied, then by the 90s of the XX century the study of the role of endogenous CTS and their regulatory effects became a popular trend in biology and physiology. The role of endogenous CTS was studied in details in the pathogenesis of cardiovascular diseases [1]. A large number of researchers have unanimously stated that endogenous CTS are very complex regulators of the development of pathological processes; however, the exact cause-and-effect relationship that would unambiguously explain the processes of synthesis, utilization, and the role of endogenous CTS has not yet been established. The effects of CTS in the frame of various regulatory schemes have been described, but the predominant number of studies was devoted to arterial hypertension, heart and renal failure, cancer [2–4], aging [5,6], and neuroinflammation. The effects of both endo- and exogenous CTS on blood cells and have been studied to a much lesser extent.