Kinetic Features of 3′–5′–Exonuclease Activity of Apurinic/Apyrimidinic Endonuclease Apn2 from Saccharomyces cerevisiae

In yeast Saccharomyces cerevisiae cells, apurinic/apyrimidinic (AP) sites are primarily repaired by base excision repair. Base excision repair is initiated by one of two AP endonucleases: Apn1 or Apn2. AP endonucleases catalyze hydrolytic cleavage of the phosphodiester backbone on the 5′ side of an AP site, thereby forming a single–strand break containing 3′–OH and 5′–dRP ends. In addition, Apn2 has 3′–phosphodiesterase activity (removing 3′–blocking groups) and 3′ → 5′ exonuclease activity (both much stronger than its AP endonuclease activity). Nonetheless, the role of the 3′–5′–exonuclease activity of Apn2 remains unclear and presumably is involved in the repair of damage containing single–strand breaks. In this work, by separating reaction products in a polyacrylamide gel and by a stopped–flow assay, we performed a kinetic analysis of the interaction of Apn2 with various model DNA substrates containing a 5′ overhang. The results allowed us to propose a mechanism for the cleaving off of nucleotides and to determine the rate of the catalytic stage of the process. It was found that dissociation of a reaction product from the enzyme active site is not a rate–limiting step in the enzymatic reaction. We determined an influence of the nature of the 3′–terminal nucleotide that can be cleaved off on the course of the enzymatic reaction. Finally, it was found that the efficiency of the enzymatic reaction is context–specific.     

Nano–Liposomes Double Loaded with Curcumin and Tetrandrine: Preparation,Characterization, Hepatotoxicity and Anti–Tumor Effects

(1) Background: Curcumin (CUR) and tetrandrine (TET) are natural compounds with various bioactivities, but have problems with low solubility, stability, and absorption rate, resulting in low bioavailability, and limited applications in food, medicine, and other fields. It is very important to improve the solubility while maintaining the high activity of drugs. Liposomes are micro–vesicles synthesized from cholesterol and lecithin. With high biocompatibility and biodegradability, liposomes can significantly improve drug solubility, efficacy, and bioavailability. (2) Methods: In this work, CUR and TET were encapsulated with nano–liposomes and g DSPE–MPEG 2000 (DP)was added as a stabilizer to achieve better physicochemical properties, biosafety, and anti–tumor effects. (3) Results: The nano–liposome (CT–DP–Lip) showed stable particle size (under 100 nm) under different conditions, high solubility, drug encapsulation efficiency (EE), loading capacity (LC), release rate in vitro, and stability. In addition, in vivo studies demonstrated CT–DP–Lip had no significant toxicity on zebrafish. Tumor cytotoxicity test showed that CT–DP–Lip had a strong inhibitory effect on a variety of cancer cells. (4) Conclusions: This work showed that nano–liposomes can significantly improve the physical and chemical properties of CUR and TET and make them safer and more efficient.     

Sequential Release of Paclitaxel and Imatinib from Core–Shell Microparticles Prepared by Coaxial Electrospray for Vaginal Therapy of Cervical Cancer

To optimize the anti-tumor efficacy of combination therapy with paclitaxel (PTX) and imatinib (IMN), we used coaxial electrospray to prepare sequential-release core–shell microparticles composed of a PTX-loaded sodium hyaluronate outer layer and an IMN-loaded PLGA core. The morphology, size distribution, drug loading, differential scanning calorimetry (DSC), Fourier transform infrared spectra (FTIR), in vitro release, PLGA degradation, cellular growth inhibition, in vivo vaginal retention, anti-tumor efficacy, and local irritation in a murine orthotopic cervicovaginal tumor model after vaginal administration were characterized. The results show that such core–shell microparticles were of spherical appearance, with an average size of 14.65 μm and a significant drug-loading ratio (2.36% for PTX, 19.5% for IMN, w/w), which might benefit cytotoxicity against cervical-cancer-related TC-1 cells. The DSC curves indicate changes in the phase state of PTX and IMN after encapsulation in microparticles. The FTIR spectra show that drug and excipients are compatible with each other. The release profiles show sequential characteristics in that PTX was almost completely released in 1 h and IMN was continuously released for 7 days. These core–shell microparticles showed synergistic inhibition in the growth of TC-1 cells. Such microparticles exhibited prolonged intravaginal residence, a >90% tumor inhibitory rate, and minimal mucosal irritation after intravaginal administration. All results suggest that such microparticles potentially provide a non-invasive local chemotherapeutic delivery system for the treatment of cervical cancer by the sequential release of PTX and IMN.     

Impact of Reactive Oxygen and Nitrogen Species Produced by Plasma on Mdm2–p53 Complex

The study of protein–protein interactions is of great interest. Several early studies focused on the murine double minute 2 (Mdm2)–tumor suppressor protein p53 interactions. However, the effect of plasma treatment on Mdm2 and p53 is still absent from the literature. This study investigated the structural changes in Mdm2, p53, and the Mdm2–p53 complex before and after possible plasma oxidation through molecular dynamic (MD) simulations. MD calculation revealed that the oxidized Mdm2 bounded or unbounded showed high flexibility that might increase the availability of tumor suppressor protein p53 in plasma-treated cells. This study provides insight into Mdm2 and p53 for a better understanding of plasma oncology.     

A 3D–Predicted Structure of the Amine Oxidase Domain of Lysyl Oxidase–Like 2

Lysyl oxidase–like 2 (LOXL2) has been recognized as an attractive drug target for anti–fibrotic and anti–tumor therapies. However, the structure–based drug design of LOXL2 has been very challenging due to the lack of structural information of the catalytically–competent LOXL2. In this study; we generated a 3D–predicted structure of the C–terminal amine oxidase domain of LOXL2 containing the lysine tyrosylquinone (LTQ) cofactor from the 2.4Å crystal structure of the Zn²⁺–bound precursor (lacking LTQ; PDB:5ZE3); this was achieved by molecular modeling and molecular dynamics simulation based on our solution studies of a mature LOXL2 that is inhibited by 2–hydrazinopyridine. The overall structures of the 3D–modeled mature LOXL2 and the Zn²⁺–bound precursor are very similar (RMSD = 1.070Å), and disulfide bonds are conserved. The major difference of the mature and the precursor LOXL2 is the secondary structure of the pentapeptide (His652–Lys653–Ala654–Ser655–Phe656) containing Lys653 (the precursor residue of the LTQ cofactor). We anticipate that this peptide is flexible in solution to accommodate the conformation that enables the LTQ cofactor formation as opposed to the β–sheet observed in 5ZE3. We discuss the active site environment surrounding LTQ and Cu²⁺ of the 3D–predicted structure.     

A Titanium (IV)–Dithiophenolate Complex and Its Chitosan Nanocomposite: Their Roles towards Rat Liver Injuries In Vivo and against Human Liver Cancer Cell Lines

Titanium (IV)–dithiophenolate complex chitosan nanocomposites (DBT–CSNPs) are featured by their antibacterial activities, cytotoxicity, and capacity to bind with DNA helixes. In this study, their therapeutic effects against rat liver damage induced by carbon tetrachloride (CCl₄) and their anti-proliferative activity against human liver cancer (HepG2) cell lines were determined. Results of treatment were compared with cisplatin treatment. Markers of apoptosis, oxidative stress, liver functions, and liver histopathology were determined. The results showed that DBT–CSNPs and DBT treatments abolished liver damage induced by CCl₄ and improved liver architecture and functions. DNA fragmentation, Bax, and caspase-8 were reduced, but Bcl-2 and the Bcl-2/Bax ratios were increased. However, there was a non-significant change in the oxidative stress markers. DBT–CSNPs and DBT inhibited the proliferation of HepG2 cells by arresting cells in the G2/M phase and inducing cell death. DBT–CSNPs were more efficient than DBT. Low doses of DBT and DBT–CSNPs applied to healthy rats for 14 days had no adverse effect. DBT and DBT–CSNP treatment gave preferable results than the treatment with cisplatin. In conclusion, DBT–CSNPs and DBT have anti-apoptotic activities against liver injuries and have anti-neoplastic impacts. DBT–CSNPs are more efficient. Both compounds can be used in pharmacological fields.     

Therapeutic Effect of Melittin–dKLA Targeting Tumor-Associated Macrophages in Melanoma

Melanoma is an immunogenic tumor and a serious type of skin cancer. Tumor-associated macrophages (TAMs) express an M2-like phenotype and are involved in all stages of melanomagenesis; it is hence a promising target for cancer immunotherapy. We herein investigated whether melittin–dKLA inhibits the growth of melanoma by inducing apoptosis of M2-like macrophages. For the in vitro study, a conditioned medium of macrophages was prepared from M0, M1, or M2-differentiated THP-1 cells with and without melittin–dKLA. The affinity of melittin for M2 macrophages was studied with FITC (fluorescein isothiocyanate)-conjugated melittin. For the in vivo study, murine melanoma cells were inoculated subcutaneously in the right flank of mice, melittin–dKLA was intraperitoneally injected at 200 nmol/kg every three days, and flow cytometry analysis of TAMs was performed. Since melittin binds preferentially to M2-like macrophages, melittin–dKLA induced more caspase 3 expression and cell death in M2 macrophages compared with M0 and M1 macrophages and melanoma cells. Melittin–dKLA significantly inhibited the proliferation and migration of M2 macrophages, resulting in a decrease in melanoma tumor growth in vivo. The CD206⁺ M2-like TAMs were reduced, while the CD86⁺ M1-like TAMs were not affected. Melittin–dKLA is therapeutically effective against melanoma by inducing the apoptosis of M2-like TAMs.     

Modulation of Aptamer–Ligand-Binding by Complementary Oligonucleotides: A G-Quadruplex Anti-Ochratoxin A Aptamer Case Study

Short oligonucleotides are widely used for the construction of aptamer-based sensors and logical bioelements to modulate aptamer–ligand binding. However, relationships between the parameters (length, location of the complementary region) of oligonucleotides and their influence on aptamer–ligand interactions remain unclear. Here, we addressed this task by comparing the effects of short complementary oligonucleotides (ssDNAs) on the structure and ligand-binding ability of an aptamer and identifying ssDNAs’ features that determine these effects. Within this, the interactions between the OTA-specific G-quadruplex aptamer 1.12.2 (5′-GATCGGGTGTGGGTGGCGTAAAGGGA GCATCGGACA-3′) and 21 single-stranded DNA (ssDNA) oligonucleotides complementary to different regions of the aptamer were studied. Two sets of aptamer–ssDNA dissociation constants were obtained in the absence and in the presence of OTA by isothermal calorimetry and fluorescence anisotropy, respectively. In both sets, the binding constants depend on the number of hydrogen bonds formed in the aptamer–ssDNA complex. The ssDNAs’ having more than 23 hydrogen bonds with the aptamer have a lower aptamer dissociation constant than for aptamer–OTA interactions. The ssDNAs’ having less than 18 hydrogen bonds did not affect the aptamer–OTA affinity. The location of ssDNA’s complementary site in the aptamer affeced the kinetics of the interaction and retention of OTA-binding in aptamer–ssDNA complexes. The location of the ssDNA site in the aptamer G-quadruplex led to its unfolding. In the presence of OTA, the unfolding process was longer and takes from 20 to 70 min. The refolding in the presence of OTA was possible and depends on the length and location of the ssDNA’s complementary site. The location of the ssDNA site in the tail region led to its rapid displacement and wasn’t affecting the G-qaudruplex’s integrity. It makes the tail region more perspective for the development of ssDNA-based tools using this aptamer.     

Thermodynamics of π–π Interactions of Benzene and Phenol in Water

The π–π interaction is a major driving force that stabilizes protein assemblies during protein folding. Recent studies have additionally demonstrated its involvement in the liquid–liquid phase separation (LLPS) of intrinsically disordered proteins (IDPs). As the participating residues in IDPs are exposed to water, π–π interactions for LLPS must be modeled in water, as opposed to the interactions that are often established at the hydrophobic domains of folded proteins. Thus, we investigated the association of free energies of benzene and phenol dimers in water by integrating van der Waals (vdW)-corrected density functional theory (DFT) and DFT in classical explicit solvents (DFT-CES). By comparing the vdW-corrected DFT and DFT-CES results with high-level wavefunction calculations and experimental solvation free energies, respectively, we established the quantitative credibility of these approaches, enabling a reliable prediction of the benzene and phenol dimer association free energies in water. We discovered that solvation influences dimer association free energies, but not significantly when no direct hydrogen-bond-type interaction exists between two monomeric units, which can be explained by the enthalpy–entropy compensation. Our comprehensive computational study of the solvation effect on π–π interactions in water could help us understand the molecular-level driving mechanism underlying the IDP phase behaviors.     

Disease-Induced Modulation of Drug Transporters at the Blood–Brain Barrier Level

The blood–brain barrier (BBB) is a highly selective and restrictive semipermeable network of cells and blood vessel constituents. All components of the neurovascular unit give to the BBB its crucial and protective function, i.e., to regulate homeostasis in the central nervous system (CNS) by removing substances from the endothelial compartment and supplying the brain with nutrients and other endogenous compounds. Many transporters have been identified that play a role in maintaining BBB integrity and homeostasis. As such, the restrictive nature of the BBB provides an obstacle for drug delivery to the CNS. Nevertheless, according to their physicochemical or pharmacological properties, drugs may reach the CNS by passive diffusion or be subjected to putative influx and/or efflux through BBB membrane transporters, allowing or limiting their distribution to the CNS. Drug transporters functionally expressed on various compartments of the BBB involve numerous proteins from either the ATP-binding cassette (ABC) or the solute carrier (SLC) superfamilies. Pathophysiological stressors, age, and age-associated disorders may alter the expression level and functionality of transporter protein elements that modulate drug distribution and accumulation into the brain, namely, drug efficacy and toxicity. This review focuses and sheds light on the influence of inflammatory conditions and diseases such as Alzheimer’s disease, epilepsy, and stroke on the expression and functionality of the BBB drug transporters, the consequential modulation of drug distribution to the brain, and their impact on drug efficacy and toxicity.     

Anticoagulant Oligonucleotide–Peptide Conjugates: Identification of Thrombin Aptamer Conjugates with Improved Characteristics

Oligonucleotide–peptide conjugates (OPCs) are a promising class of biologically active compounds with proven potential for improving nucleic acid therapeutics. OPCs are commonly recognized as an efficient instrument to enhance the cellular delivery of therapeutic nucleic acids. In addition to this application field, OPCs have an as yet unexplored potential for the post-SELEX optimization of DNA aptamers. In this paper, we report the preparation of designer thrombin aptamer OPCs with peptide side chains anchored to a particular thymidine residue of the aptamer. The current conjugation strategy utilizes unmodified short peptides and support-bound protected oligonucleotides with activated carboxyl functionality at the T3 thymine nucleobase. The respective modification of the oligonucleotide strand was implemented using N3-derivatized thymidine phosphoramidite. Aptamer OPCs retained the G-quadruplex architecture of the parent DNA structure and showed minor to moderate stabilization. In a series of five OPCs, conjugates bearing T3–Ser–Phe–Asn (SFN) or T3–Tyr–Trp–Asn (YWN) side chains exhibited considerably improved anticoagulant characteristics. Molecular dynamics studies of the aptamer OPC complexes with thrombin revealed the roles of the amino acid nature and sequence in the peptide subunit in modulating the anticoagulant activity.     

The Protective Effects on Ischemia–Reperfusion Injury Mechanisms of the Thoracic Aorta in Daurian Ground Squirrels (Spermophilus dauricus) over the Torpor–Arousal Cycle of Hibernation

Hibernators are a natural model of vascular ischemia–reperfusion injury; however, the protective mechanisms involved in dealing with such an injury over the torpor–arousal cycle are unclear. The present study aimed to clarify the changes in the thoracic aorta and serum in summer-active (SA), late-torpor (LT) and interbout-arousal (IBA) Daurian ground squirrels (Spermophilus dauricus). The results show that total antioxidant capacity (TAC) was unchanged, but malondialdehyde (MDA), hydrogen peroxide (H₂O₂), interleukin-1β (IL-1β) and tumor necrosis factor α (TNFα) were significantly increased for the LT group, whereas the levels of superoxide dismutase (SOD) and interleukin-10 (IL-10) were significantly reduced in the LT group as compared with the SA group. Moreover, the levels of MDA and IL-1β were significantly reduced, whereas SOD and IL-10 were significantly increased in the IBA group as compared with the SA group. In addition, the lumen area of the thoracic aorta and the expression of the smooth muscle cells (SMCs) contractile marker protein 22α (SM22α) were significantly reduced, whereas the protein expression of the synthetic marker proteins osteopontin (OPN), vimentin (VIM) and proliferating cell nuclear antigen (PCNA) were significantly increased in the LT group as compared with the SA group. Furthermore, the smooth muscle layer of the thoracic aorta was significantly thickened, and PCNA protein expression was significantly reduced in the IBA group as compared with the SA group. The contractile marker proteins SM22α and synthetic marker protein VIM underwent significant localization changes in both LT and IBA groups, with localization of the contractile marker protein α-smooth muscle actin (αSMA) changing only in the IBA group as compared with the SA group. In tunica intima, the serum levels of heparin sulfate (HS) and syndecan-1 (Sy-1) in the LT group were significantly reduced, but the serum level of HS in the IBA group increased significantly as compared with the SA group. Protein expression and localization of endothelial nitric oxide synthase (eNOS) was unchanged in the three groups. In summary, the decrease in reactive oxygen species (ROS) and pro-inflammatory factors and increase in SOD and anti-inflammatory factors during the IBA period induced controlled phenotypic switching of thoracic aortic SMCs and restoration of endothelial permeability to resist ischemic and hypoxic injury during torpor of Daurian ground squirrels.     

Extracellular Prion Protein Aggregates in Nine Gerstmann–Sträussler–Scheinker Syndrome Subjects with Mutation P102L: A Micromorphological Study and Comparison with Literature Data

Gerstmann–Sträussler–Scheinker syndrome (GSS) is a hereditary neurodegenerative disease characterized by extracellular aggregations of pathological prion protein (PrP) forming characteristic plaques. Our study aimed to evaluate the micromorphology and protein composition of these plaques in relation to age, disease duration, and co-expression of other pathogenic proteins related to other neurodegenerations. Hippocampal regions of nine clinically, neuropathologically, and genetically confirmed GSS subjects were investigated using immunohistochemistry and multichannel confocal fluorescent microscopy. Most pathognomic prion protein plaques were small (2–10 µm), condensed, globous, and did not contain any of the other investigated proteinaceous components, particularly dystrophic neurites. Equally rare (in two cases out of nine) were plaques over 50 µm having predominantly fibrillar structure and exhibit the presence of dystrophic neuritic structures; in one case, the plaques also included bulbous dystrophic neurites. Co-expression with hyperphosphorylated protein tau protein or amyloid beta-peptide (Aβ) in GSS PrP plaques is generally a rare observation, even in cases with comorbid neuropathology. The dominant picture of the GSS brain is small, condensed plaques, often multicentric, while presence of dystrophic neuritic changes accumulating hyperphosphorylated protein tau or Aβ in the PrP plaques are rare and, thus, their presence probably constitutes a trivial observation without any relationship to GSS development and progression.     

The Anti-Tumor Activity of the NEDD8 Inhibitor Pevonedistat in Neuroblastoma

Pevonedistat is a neddylation inhibitor that blocks proteasomal degradation of cullin–RING ligase (CRL) proteins involved in the degradation of short-lived regulatory proteins, including those involved with cell-cycle regulation. We determined the sensitivity and mechanism of action of pevonedistat cytotoxicity in neuroblastoma. Pevonedistat cytotoxicity was assessed using cell viability assays and apoptosis. We examined mechanisms of action using flow cytometry, bromodeoxyuridine (BrDU) and immunoblots. Orthotopic mouse xenografts of human neuroblastoma were generated to assess in vivo anti-tumor activity. Neuroblastoma cell lines were very sensitive to pevonedistat (IC50 136–400 nM). The mechanism of pevonedistat cytotoxicity depended on p53 status. Neuroblastoma cells with mutant (p53^MUT) or reduced levels of wild-type p53 (p53si-p53) underwent G2-M cell-cycle arrest with rereplication, whereas p53 wild-type (p53^WT) cell lines underwent G0-G1 cell-cycle arrest and apoptosis. In orthotopic neuroblastoma models, pevonedistat decreased tumor weight independent of p53 status. Control mice had an average tumor weight of 1.6 mg + 0.8 mg versus 0.5 mg + 0.4 mg (p < 0.05) in mice treated with pevonedistat. The mechanism of action of pevonedistat in neuroblastoma cell lines in vitro appears p53 dependent. However, in vivo studies using mouse neuroblastoma orthotopic models showed a significant decrease in tumor weight following pevonedistat treatment independent of the p53 status. Novel chemotherapy agents, such as the NEDD8-activating enzyme (NAE) inhibitor pevonedistat, deserve further study in the treatment of neuroblastoma.     

Golden and Silver–Golden Chitosan Hydrogels and Fabrics Modified with Golden Chitosan Hydrogels

Golden and silver–golden chitosan hydrogels and hydrogel-modified textiles of potential biomedical applications are investigated in this work. The hydrogels are formed by reactions of chitosan with HAuCl₄·xH₂O. For above the critical concentration of chitosan (c*), chitosan–Au hydrogels were prepared. For chitosan concentrations lower than c*, chitosan–Au nano- and microgels were formed. To characterise chitosan–Au structures, sol–gel analysis, UV–Vis spectrophotometry and dynamic light scattering were performed. Au concentration in the hydrogels was determined by the flame atomic absorption spectrophotometry. Colloidal chitosan–Au solutions were used for the modification of fabrics. The Au content in the modified fabrics was quantified by inductively coupled plasma mass spectrometry technique. Scanning electron microscopy with energy dispersion X-ray spectrometer was used to analyse the samples. Reflectance spectrophotometry was applied to examine the colour of the fabrics. The formation of chitosan–Au–Ag hydrogels by the competitive reaction of Au and Ag ions with the chitosan macromolecules is reported.     

Role of the Renin–Angiotensin–Aldosterone and Kinin–Kallikrein Systems in the Cardiovascular Complications of COVID-19 and Long COVID

Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) is the virus responsible for the COVID-19 pandemic. Patients may present as asymptomatic or demonstrate mild to severe and life-threatening symptoms. Although COVID-19 has a respiratory focus, there are major cardiovascular complications (CVCs) associated with infection. The reported CVCs include myocarditis, heart failure, arrhythmias, thromboembolism and blood pressure abnormalities. These occur, in part, because of dysregulation of the Renin–Angiotensin–Aldosterone System (RAAS) and Kinin–Kallikrein System (KKS). A major route by which SARS-CoV-2 gains cellular entry is via the docking of the viral spike (S) protein to the membrane-bound angiotensin converting enzyme 2 (ACE2). The roles of ACE2 within the cardiovascular and immune systems are vital to ensure homeostasis. The key routes for the development of CVCs and the recently described long COVID have been hypothesised as the direct consequences of the viral S protein/ACE2 axis, downregulation of ACE2 and the resulting damage inflicted by the immune response. Here, we review the impact of COVID-19 on the cardiovascular system, the mechanisms by which dysregulation of the RAAS and KKS can occur following virus infection and the future implications for pharmacological therapies.     

Binding of Amyloid β(1–42)-Calmodulin Complexes to Plasma Membrane Lipid Rafts in Cerebellar Granule Neurons Alters Resting Cytosolic Calcium Homeostasis

Lipid rafts are a primary target in studies of amyloid β (Aβ) cytotoxicity in neurons. Exogenous Aβ peptides bind to lipid rafts, which in turn play a key role in Aβ uptake, leading to the formation of neurotoxic intracellular Aβ aggregates. On the other hand, dysregulation of intracellular calcium homeostasis in neurons has been observed in Alzheimer’s disease (AD). In a previous work, we showed that Aβ(1–42), the prevalent Aβ peptide found in the amyloid plaques of AD patients, binds with high affinity to purified calmodulin (CaM), with a dissociation constant ≈1 nM. In this work, to experimentally assess the Aβ(1–42) binding capacity to intracellular CaM, we used primary cultures of mature cerebellar granule neurons (CGN) as a neuronal model. Our results showed a large complexation of submicromolar concentrations of Aβ(1–42) dimers by CaM in CGN, up to 120 ± 13 picomoles of Aβ(1–42) /2.5 × 10⁶ cells. Using fluorescence microscopy imaging, we showed an extensive co-localization of CaM and Aβ(1–42) in lipid rafts in CGN stained with up to 100 picomoles of Aβ(1–42)-HiLyteTM-Fluor555 monomers. Intracellular Aβ(1–42) concentration in this range was achieved by 2 h incubation of CGN with 2 μM Aβ(1–42), and this treatment lowered the resting cytosolic calcium of mature CGN in partially depolarizing 25 mM potassium medium. We conclude that the primary cause of the resting cytosolic calcium decrease is the inhibition of L-type calcium channels of CGN by Aβ(1–42) dimers, whose activity is inhibited by CaM:Aβ(1–42) complexes bound to lipid rafts.     

Genotoxicological Characterization of (±)cis-4,4′-DMAR and (±)trans-4,4′-DMAR and Their Association

The novel psychoactive substance (NPS) 4-Methyl-5-(4-methylphenyl)-4,5-dihydroxazol-2-amine (4,4′-DMAR) shows psychostimulant activity. Data on the acute toxicity of 4,4′-DMAR are becoming increasingly available, yet the long-term effects are still almost unknown. In particular, no data on genotoxicity are available. Therefore, the aim of the present study was to evaluate its genotoxic potential using the “In Vitro Mammalian Cell Micronucleus Test” (MNvit) on (±)cis-4,4′-DMAR and (±)trans-4,4′-DMAR and their associations. The analyses were conducted in vitro on human TK6 cells. To select suitable concentrations for MNvit, we preliminarily evaluated cytotoxicity and apoptosis. All endpoints were analysed by flow cytometry. The results reveal the two racemates’ opposite behaviours: (±)cis-4,4′-DMAR shows a statistically significant increase in micronuclei (MNi) frequency that (±)trans-4,4′-DMAR is completely incapable of. This contrast confirms the well-known possibility of observing opposite biological effects of the cis- and trans- isomers of a compound, and it highlights the importance of testing single NPSs that show even small differences in structure or conformation. The genotoxic capacity demonstrated stresses an additional alarming toxicological concern related to this NPS. Moreover, the co-treatments indicate that consuming both racemates will magnify the genotoxic effect, an aspect to consider given the unpredictability of illicit drug composition.     

Smart Lipid–Polysaccharide Nanoparticles for Targeted Delivery of Doxorubicin to Breast Cancer Cells

In this study, actively-targeted (CD44-receptors) and dual stimuli (pH/redox)-responsive lipid–polymer nanoparticles were proposed as a delivery vehicle of doxorubicin hydrochloride in triple negative breast cancer cell lines. A phosphatidylcholine lipid film was hydrated with a solution of oxidized hyaluronic acid and doxorubicin, chosen as model drug, followed by a crosslinking reaction with cystamine hydrochloride. The obtained spherical nanoparticles (mean diameter of 30 nm) were found to be efficiently internalized in cancer cells by a receptor-mediated endocytosis process, and to modulate the drug release depending on the pH and redox potential of the surrounding medium. In vitro cytotoxicity assays demonstrated the safety and efficacy of the nanoparticles in enhancing the cytotoxic effect of the free anticancer drug, with the IC₅₀ values being reduced by two and three times in MDA-MB-468 and MDA-MB-231, respectively. The combination of self-assembled phospholipid molecules with a polysaccharide counterpart acting as receptor ligand, and stimuli-responsive chemical moieties, was carried out on smart multifunctional nanoparticles able to actively target breast cancer cells and improve the in vitro anticancer activity of doxorubicin.     

Ligand-Dependent and Ligand-Independent Effects of Ephrin-B2–EphB4 Signaling in Melanoma Metastatic Spine Disease

Tumor–endothelial cell interactions represent an essential mechanism in spinal metastasis. Ephrin-B2–EphB4 communication induces tumor cell repulsion from the endothelium in metastatic melanoma, reducing spinal bone metastasis formation. To shed further light on the Ephrin-B2–EphB4 signaling mechanism, we researched the effects of pharmacological EphB4 receptor stimulation and inhibition in a ligand-dependent/independent context. We chose a preventative and a post-diagnostic therapeutic window. EphB4 stimulation during tumor cell seeding led to an increase in spinal metastatic loci and number of disseminated melanoma cells, as well as earlier locomotion deficits in the presence of endothelial Ephrin-B2. In the absence of endothelial Ephrin-B2, reduction of metastatic loci with a later manifestation of locomotion deficits occurred. Thus, EphB4 receptor stimulation affects metastatic dissemination depending on the presence/absence of endothelial Ephrin-B2. After the manifestation of solid metastasis, EphB4 kinase inhibition resulted in significantly earlier manifestation of locomotion deficits in the presence of the ligand. No post-diagnostic treatment effect was found in the absence of endothelial Ephrin-B2. For solid metastasis treatment, EphB4 kinase inhibition induced prometastatic effects in the presence of endothelial Ephrin-B2. In the absence of endothelial Ephrin-B2, both therapies showed no effect on the growth of solid metastasis.