Importance of Translation–Replication Balance for Efficient Replication by the Self‐Encoded Replicase

In all living systems, the genetic information is replicated by the self‐encoded replicase (Rep); this can be said to be a self‐encoding system. Recently, we constructed a self‐encoding system in liposomes as an artificial cell model, consisting of a reconstituted translation system and an RNA encoding the catalytic subunit of Qβ Rep and the RNA was replicated by the self‐encoded Rep produced by the translation reaction. In this system, both the ribosome (Rib) and Rep bind to the same RNA for translation and replication, respectively. Thus, there could be a dilemma: effective RNA replication requires high levels of Rep translation, but excessive translation in turn inhibits replication. Herein, we actually observed the competition between the Rib and Rep, and evaluated the effect for RNA replication by constructing a kinetic model that quantitatively explained the behavior of the self‐encoding system. Both the experimental and theoretical results consistently indicated that the balance between translation and replication is critical for an efficient self‐encoded system, and we determined the optimum balance.     

Effect of Liposome Size on Internal RNA Replication Coupled with Replicase Translation

Cell membranes inhibit the diffusion of intracellular materials, and compartment size can strongly affect the intracellular biochemical reactions. To assess the effect of the size of microcompartments on intracellular reactions, we constructed a primitive cell model consisting of giant liposomes and a translation‐coupled RNA replication (TcRR) system. The RNA was replicated by Qβ replicase, which was translated from the RNA in giant liposomes encapsulating the cell‐free translation system. A reporter RNA encoding the antisense strand of β‐glucuronidase was introduced into the system to yield a TcRR read‐out (green fluorescence). We demonstrate that TcRR was hardly detectable in larger liposomes (230 fL) but was more effective in smaller (7.7 fL) liposomes. Our experimental and theoretical results show that smaller microcompartments considerably enhance TcRR because the synthesized molecules, such as RNA and replicases, are more concentrated in smaller liposomes.     

α‐Complementation in an Artificial Genome Replication System in Liposomes

Genome size is considered one of the limiting factors for the replication of primitive life forms. However, the relationship between genome size and replication efficiency has not been tested experimentally. In this study, we examined the effect of genome size on genome replication by using an artificial cell model: a self‐replicating RNA genome encapsulated in a liposome. For the reduced genome size we used α‐complementation of the lacZ gene. We first characterized α‐complementation in the purified translation system and then applied α‐complementation to the genome replication system. The reduction in the genome size together with the addition of ω‐fragment increased the replication efficiency approximately eightfold. This result provides experimental evidence that genome size can be a limiting factor for primitive self‐replication systems; it also implies that this artificial cell model could be a useful experimental model to identify possible mechanisms of genome enlargement.     

Retention of Eucalyptol,a Natural Volatile Insecticide,in Delivery Systems Based on Hydroxypropyl‐β‐Cyclodextrin and Liposomes

Eucalyptol (Euc) is a natural monoterpene with insecticide effects. Being highly volatile and sensitive to ambient conditions, its encapsulation would enlarge its application. Euc‐loaded conventional liposomes (CL), cyclodextrin/drug inclusion complex, and drug‐in‐cyclodextrin‐in‐liposomes (DCL) are prepared to protect Euc from degradation, reduce its evaporation, and provide its controlled release. The liposomal suspension is freeze‐dried using hydroxypropyl‐β‐cyclodextrin (HP‐β‐CD) as cryoprotectant. The liposomes are characterized before and after freeze‐drying. The effect of Euc on the fluidity of liposomal membrane is also examined. A release study of Euc from delivery systems, in powder and reconstituted forms, is performed by multiple head extraction at 60 °C after 6 months of storage at 4 °C. CL and DCL suspensions are homogeneous, show nanometric vesicles size, spherical shape, and negative surface charge before and after freeze‐drying. Moreover, HP‐β‐CD does not affect the fluidity of liposomes. CL formulations present a weak encapsulation for Euc. The loading capacity of eucalyptol in DCL is 38 times higher than that in CL formulation. In addition, freeze‐dried DCL and HP‐β‐CD/Euc inclusion complex show a higher retention of eucalyptol than CL delivery system. Both carrier systems HP‐β‐CD/Euc and Euc‐loaded DCL decrease Euc evaporation and improve its retention. Practical Applications: Eucalyptol is a natural insecticide. It is highly volatile and poorly soluble in water. To enlarge its application, its encapsulation in three delivery systems (conventional liposomes, cyclodextrin/drug inclusion complex, combined system composed of cyclodextrin inclusion complex and liposome) is studied. In this paper it is proved that cyclodextrin/eucalyptol inclusion complex and eucalyptol‐in‐cyclodextrin‐in‐liposome are effective delivery systems for encalyptol encapsulation, retention, and release.     

Unbound DHA causes a high blank value in β‐oxidation assay: a concern for in vitro studies

The information on binding capacity of different fatty acids (FAs) to albumin is incomplete, however, in the majority of in vitro experiments, FAs and albumin were simply mixed and their affinity believed to be complete. In this study, seven [1‐¹⁴C] FAs were mixed with albumin and assayed for β‐oxidation in rat liver homogenates. In the process of identifying the radioactive background of control assay by LCMS/MS, the results indicated different binding capacity of FAs to albumin. The percentage of unbound FAs recovered in clarified acidic solution was lower than 2% with 16:0 and 18:1n‐9, nearly 5% with EPA, 7% with 18:2n‐6, 18:3n‐3 and 20:4n‐6, and surprisingly high to 41% with DHA. Various FA/albumin molar ratios as well as different types of albumin only marginally affected these data. Thus, the big mass of unbound free DHA led to a high blank value, which is 60 times higher than the real value in the procedure of β‐oxidation measurement in vitro. In the design of future FA research in vitro, the binding capacity of FA to albumin or other proteins must be considered, especially for DHA research.     

Role of RNA Motifs in RNA Interaction with Membrane Lipid Rafts: Implications for Therapeutic Applications of Exosomal RNAs

RNA motifs may promote interactions with exosomes (EXO-motifs) and lipid rafts (RAFT-motifs) that are enriched in exosomal membranes. These interactions can promote selective RNA loading into exosomes. We quantified the affinity between RNA aptamers containing various EXO- and RAFT-motifs and membrane lipid rafts in a liposome model of exosomes by determining the dissociation constants. Analysis of the secondary structure of RNA molecules provided data about the possible location of EXO- and RAFT-motifs within the RNA structure. The affinity of RNAs containing RAFT-motifs (UUGU, UCCC, CUCC, CCCU) and some EXO-motifs (CCCU, UCCU) to rafted liposomes is higher in comparison to aptamers without these motifs, suggesting direct RNA-exosome interaction. We have confirmed these results through the determination of the dissociation constant values of exosome-RNA aptamer complexes. RNAs containing EXO-motifs GGAG or UGAG have substantially lower affinity to lipid rafts, suggesting indirect RNA-exosome interaction via RNA binding proteins. Bioinformatics analysis revealed RNA aptamers containing both raft- and miRNA-binding motifs and involvement of raft-binding motifs UCCCU and CUCCC. A strategy is proposed for using functional RNA aptamers (fRNAa) containing both RAFT-motif and a therapeutic motif (e.g., miRNA inhibitor) to selectively introduce RNAs into exosomes for fRNAa delivery to target cells for personalized therapy.     

Hispolon Cyclodextrin Complexes and Their Inclusion in Liposomes for Enhanced Delivery in Melanoma Cell Lines

Hispolon, a phenolic pigment isolated from the mushroom species Phellinus linteus, has been investigated for anti-inflammatory, antioxidant, and anticancer properties; however, low solubility and poor bioavailability have limited its potential clinical translation. In this study, the inclusion complex of hispolon with Sulfobutylether-β-cyclodextrin (SBEβCD) was characterized, and the Hispolon-SBEβCD Complex (HSC) was included within the sterically stabilized liposomes (SL) to further investigate its anticancer activity against melanoma cell lines. The HSC-trapped-Liposome (HSC-SL) formulation was investigated for its sustained drug delivery and enhanced cytotoxicity. The inclusion complex in the solid=state was confirmed by a Job’s plot analysis, molecular modeling, differential scanning calorimetry (DSC), Fourier transform infrared spectroscopy (FTIR), Proton nuclear magnetic resonance (NMR) spectroscopy, and scanning electron microscopy (SEM). The HSC-SL showed no appreciable deviation in size (<150 nm) and polydispersity index (<0.2) and improved drug encapsulation efficiency (>90%) as compared to control hispolon liposomes. Individually incorporated hispolon and SBEβCD in the liposomes (H-CD-SL) was not significant in loading the drug in the liposomes, compared to HSC-SL, as a substantial amount of free drug was separated during dialysis. The HSC-SL formulation showed a sustained release compared to hispolon liposomes (H-SLs) and Hispolon-SBEβCD liposomes (H-CD-SLs). The anticancer activity on melanoma cell lines (B16BL6) of HSC and HSC-SL was higher than in H-CD-SL and hispolon solution. These findings suggest that HSC inclusion in the HSC-SL liposomes stands out as a potential formulation approach for enhancing drug loading, encapsulation, and chemotherapeutic efficiency of hispolon and similar water insoluble drug molecules.     

Inhibitory Effect of N-Acyl Dopamines on IgE-Mediated Allergic Response in RBL-2H3 Cells

Recently, endogenous N-acyl dopamines have been found to show anti-inflammatory and immunomodulatory activities. However, the effect of the N-acyl dopamines on allergic responses was not reported. In this study, we investigated whether N-acyl dopamines might inhibit immunoglobulin E-mediated degranulation in RBL-2H3 cells. When RBL-2H3 cells were exposed to palmitoyl dopamine (NP-DA), oleoyl dopamine (NO-DA) or arachidonoyl dopamine (NA-DA) at micromolar levels, all these compounds significantly inhibited the release of β-hexosaminidase, a marker of degranulation, as well as tumor necrosis factor (TNF)-α. In comparison, NP-DA, potently suppressing the release of β-hexosaminidase (IC₅₀, 3.5 μM) and TNF-α (IC₅₀, 2.2 μM), was more potent than NO-DA or NA-DA. Additionally, NP-DA markedly suppressed the formation of prostaglandin E₂, prostaglandin D₂ and leukotriene C₄, corresponding to pro-inflammatory lipid mediators in asthma. In the mechanistic analyses, where the effect of NP-DA on the FcεRI cascade was examined, NP-DA significantly inhibited the phosphorylation and expression of Syk, but not Lyn. And, NP-DA also suppressed phosphorylation of ERK1/2 and Akt. Further, NP-DA decreased the phosphorylation of cPLA₂ and 5-lipoxygenase (5-LO), but not cyclooxygenase-2 (COX-2). Based on these results, it is suggested that NP-DA exert anti-allergic effect on allergic response through suppressing the activation of Syk, ERK1/2, Akt, cPLA₂ and 5-LO. Besides, a strong inhibition of COX-2 activity by NP-DA may be additional mechanism for its anti-allergic action. Such an anti-allergic action of N-acyl dopamines may contribute to further information about biological functions of N-acyl dopamines.     

Phototoxicity Testing of Polycyclic Aromatic Hydrocarbons (PAH) in Mammalian Cells in vitro

The cytotoxicity of various PAH was determined in V79 Chinese hamster fibroblasts in the absence and presence of a solar simulator, i.e. a light source emitting visible light, including UV-A and UV-B components. The range of cytotoxic concentrations of the different PAH in the absence of light varied by a factor of about 100. The cytotoxicity of some PAH was weakly, that of others was markedly increased in the presence of light, indicating a clear phototoxic effect. The range of the phototoxic concentrations in the presence of light was much wider (about 5000-fold) than in its absence. The greatest phototoxic potential was determined for larger, as opposed to smaller PAH. Methylation caused a decrease of the phototoxicity of PAH in comparison to their unmethylated parent compounds.     

Liposome‐Based in Vitro Evolution of Aminoacyl‐tRNA Synthetase for Enhanced Pyrrolysine Derivative Incorporation

Methanosarcina species pyrrolysyl‐tRNA synthetase (PylRS) attaches Pyl to its cognate amber suppressor tRNA. The introduction of two mutations (Y384F and Y306A) into PylRS was previously shown to generate a mutant, designated LysZ‐RS, that was able to attach N‐benzyloxycarbonyl‐L ‐lysine (LysZ) to its cognate tRNA. Despite the potential of LysZ derivatives, further LysZ‐RS engineering has not been performed; consequently, we aimed to generate LysZ‐RS mutants with improved LysZ incorporation activity through in vitro directed evolution. Using a liposome‐based in vitro compartmentalization (IVC) approach, we screened a randomly mutagenized gene library of LysZ‐RS and obtained a mutant that showed increased LysZ incorporation activity both in vitro and in vivo. The ease and high flexibility of liposome‐based IVC should enable the evolution of not only LysZ‐RS that can attach various LysZ derivatives but also of other enzymes involved in protein translation.     

SIRT2 Is Critical for Sheep Oocyte Maturation through Regulating Function of Surrounding Granulosa Cells

Oocyte in vitro maturation is crucial for in vitro embryo production technology, which provides oocytes resources for in vitro fertilization and somatic cell nuclear transfer. Previous studies proved that SIRT2, a member of the sirtuin family, plays a role in oocyte meiosis, but its role in sheep oocyte maturation and its regulating mechanism remains unknown. Firstly, we confirmed the role of Sirt2 in sheep oocytes maturation by supplementation of SIRT2 inhibitor and activator. To further explore the specific mechanism, we performed knockdown of Sirt2 in granulosa cells and then cocultured them with oocytes. Moreover, we determined the effects of Sirt2 on granulosa cell oxidative apoptosis, cell migration, and diffusion, and examined its effects on granulosa cell mitochondrial function, mitophagy, and steroid hormone levels. The results showed that supplementation of SIRT2 inhibitor decreased the oocytes maturation rate (69.28% ± 1.28 vs. 45.74% ± 4.74, p < 0.05), while resveratrol, a SIRT2 activator, increased its maturation rate (67.44% ± 1.68 vs. 78.52 ± 1.28, p < 0.05). Knockdown of Sirt2 in sheep granulosa cells also reduced the oocytes maturation rate (47.98% ± 1.43 vs. 33.60% ± 1.77, p < 0.05), and led to decreased cell migration and expansion ability, oxidative apoptosis, abnormal mitochondrial gene expression, decreased mitochondrial membrane potential and ATP level, and increased mitophagy level. Overexpression of Sirt2 improved mitochondrial membrane potential and ATP level and improved mitochondrial function. Furthermore, we found that Sirt2 knockdown in granulosa cells promotes the secretion of P₄ through regulating p-ERK1/2. In conclusion the present study showed that SIRT2 is critical for sheep oocyte maturation through regulating the function of ovarian granulosa cells, especially affecting its mitochondrial function.     

Development of a ligation-based impedimetric DNA sensor for single-nucleotide polymorphism associated with metabolic syndrome

Single-nucleotide polymorphism (SNP) detection is becoming important in molecular diagnostics, clinical assay, and novel drug development. Electrochemical methods are well suited for the DNA diagnostics system. Since electrochemical reactions directly emit an electronic signal, expensive signal transduction equipment is not required. We describe the development of a novel DNA sensor that utilizes impedance spectroscopy and DNA ligation reaction on a gold electrode. Impedance spectroscopy enables label-free detection and is nondestructive and useful in equivalent circuit models for interpretation on an electrode surface, whereas from the ligation reaction, the specificity is derived by the allele-specific oligonucleotide of the capture probe on immobilized gold electrode. In other words, DNA diagnostics system using the combination of impedance spectroscopy and ligation reaction is simple, rapid, and allele specific. In this report, we have described a ligation-based impedimetric DNA sensor and the analysis of Trp64Arg polymorphism in the beta3-adrenergic receptor gene (ADRβ3).     

Liposomal Formulations of a New Zinc(II) Complex Exhibiting High Therapeutic Potential in a Murine Colon Cancer Model

Colorectal cancer is the second leading cause of cancer-related mortality. Many current therapies rely on chemotherapeutic agents with poor specificity for tumor cells. The clinical success of cisplatin has prompted the research and design of a huge number of metal-based complexes as potential chemotherapeutic agents. In this study, two zinc(II) complexes, [ZnL₂] and [ZnL(AcO)], where AcO is acetate and L is an organic compound combining 8-hydroxyquinoline and a benzothiazole moiety, were developed and characterized. Analytical and spectroscopic studies, namely, NMR, FTIR, and UV-Vis allowed us to establish the complexes’ structures, demonstrating the ligand-binding versatility: tetradentate in [ZnL(AcO)] and bidentate in [ZnL₂]. Complexes were screened in vitro using murine and human colon cancer cells cultured in 2D and 3D settings. In 2D cells, the IC₅₀ values were <22 µM, while in 3D settings, much higher concentrations were required. [ZnL(AcO)] displayed more suitable antiproliferative properties than [ZnL₂] and was chosen for further studies. Moreover, based on the weak selectivity of the zinc-based complex towards cancer cell lines in comparison to the non-tumorigenic cell line, its incorporation in long-blood-circulating liposomes was performed, aiming to improve its targetability. The resultant optimized liposomal nanoformulation presented an I.E. of 76% with a mean size under 130 nm and a neutral surface charge and released the metal complex in a pH-dependent manner. The antiproliferative properties of [ZnL(AcO)] were maintained after liposomal incorporation. Preliminary safety assays were carried out through hemolytic activity that never surpassed 2% for the free and liposomal forms of [ZnL(AcO)]. Finally, in a syngeneic murine colon cancer mouse model, while free [ZnL(AcO)] was not able to impair tumor progression, the respective liposomal nanoformulation was able to reduce the relative tumor volume in the same manner as the positive control 5-fluorouracil but, most importantly, using a dosage that was 3-fold lower. Overall, our results show that liposomes were able to solve the solubility issues of the new metal-based complex and target it to tumor sites.