As a nature-derived lipid, stearic acid (SA) has been widely recognized as a safe pharmaceutical excipient. One of the most potent application of SA in pharmaceutical aspect is its feasibility for constructing a versatile drug delivery system with translational potential, solid lipid microparticles (SLM). In this work, the formulation of SA-based SLM was screened and optimized on gram-scale (> 2 g), so as to boost the industrialization of related products. Melt-sonication method was employed for SLM production. Four critical parameters were set at five levels, viz. Lipid amount 2.0~4.0 g, sonication time 10~50 min, aqueous solution volume 60~300 mL and surfactant concentration 0~1.00% (W/V), and SLM were accordingly prepared. Reproducibility and yield were two criteria for formulation screening, and a high reproducibility and yield would be satisfactory. Herein, reproducibility and yield was determined by size deviation (reflexed by an index C) and recovered weight (reflexed by an index Y), respectively. To be specific, C = RSD²_Size + RSD²_PDI and Y = m_a- m_b, where RSD_Size, RSD_PDI, m_a and m_b represented relative standard deviation of particle size, relative standard deviation of polydispersity index, initial weight and obtained weight, respectively. The results showed that lipid amount 2.5 g, sonication time 30 min, aqueous solution volume 120 mL and surfactant concentration 0.75% (W/V) established the optimal formulation. We believe that this work can provide useful information for the (pre-)industrial-scale development of SA-based SLM.

The term ferroptosis refers to a peculiar type of programmed cell death (PCD) showing characteristic features that differentiate it from other historically well-known types of cellular death such as apoptosis, autophagy, necrosis and necroptosis. Ferroptosis is mainly characterized by extensive iron-dependent lipid peroxidation and mitochondrial dysfunction, together with the rounded morphology of the cell undergoing ferroptotic death. Recently, ferroptosis has been suggested as a potential new strategy for the treatment of several cancers, including breast cancer (BC). In particular, among the BC subtypes, triple negative breast cancer (TNBC) is considered the most aggressive, and conventional drugs fail to provide long-term efficacy. Our study’s purpose was to investigate the mechanism of ferroptosis in breast cancer cell lines and reveal the significance of heme oxygenase (HO) modulation in the process. HO’s effect on BC was evaluated by MTT tests, gene silencing, Western blot analysis, and measurement of reactive oxygen species (ROS), glutathione (GSH) and lipid hydroperoxide (LOOH) levels. In order to assess HO’s implication, different approaches were exploited, using two distinct HO-1 inducers (hemin and curcumin), a well-known HO inhibitor (SnMP) and a selective HO-2 inhibitor. The data obtained showed HO’s contribution to the onset of ferroptosis; in particular, HO-1 induction seemed to accelerate the process. Moreover, our results suggest a potential role of HO-2 in erastin-induced ferroptosis. In view of the above, HO modulation in ferroptosis can offer a novel approach for breast cancer treatment.

A method is described for high-quality protein crystal solution growth with the help of localized action of a thermal control field. Two techniques for the nucleation and growth of single crystals of biological macromolecules have been proposed. The first one utilizes a very slow temperature shift at a capillary point where the crystal is to be grown. This allows to suppress an undesirable multiple nucleation. The second technique includes several local rapid temperature changes (a temperature “shock”) forcing the nucleation at the given point.

A mathematical model has been developed and computational investigation has been performed of the processes of protein crystallization from a homogeneous aqueous solution in the crystallization volume. The mathematical model describes crystal nucleation and growth depending on the local supersaturation and temperature as well as heat-and-mass exchange within the entire volume of the solution including the protein crystals.

The temperature was shown to be a factor, capable to initiate and drive the crystallization, and also to influence the nucleation stage and, hence, the protein crystal morphology. This may be useful for obtaining good quality single crystals of biological macromolecules. One or the other technique may be chosen. The stronger the dependence of protein solubility against the temperature, the better chances one has to succeed with any of the approaches, especially with the first one.

The mathematical model developed describes the process of nucleation and growth of protein crystals from solution under the control action of a thermal field based on an intermediate phase concept, the intermediate phase consisting of a mixture of solid and liquid phase fractions. This model was used to calculate an experiment on growing a protein crystal from a homogeneous aqueous solution, with the process of crystal nucleation and growth being acted upon by the precipitant and the thermal field. The calculations showed that this model is adequate to the processes being modeled and can be used for parametric investigations and predictive calculations of protein crystallization processes under thermal control field conditions both under terrestrial and space conditions.

These techniques were successfully tested while growing single crystals of lysozyme, xylanase and human serum albumin (HSA) respectively.

Bax is a major player in the mitochondrial pathway of apoptosis, by permeabilizing the Outer Mitochondrial Membrane (OMM) to various apoptogenic factors, including cytochrome c. In order to get further insight into the structure and function of Bax when it is inserted in the OMM, we attempted to reconstitute Bax in nanodiscs. Cell-free protein synthesis in the presence of nanodiscs did not allow to obtain Bax-containing nanodiscs, but it provided a simple way to purify full-length Bax without any tag. Purified wild-type Bax (BaxWT) and a constitutively active mutant (BaxP168A) displayed structural and functionnal properties that were in line with previous characterizations following their expression in yeast and human cells followed by their reconstitution into liposomes, showing that the mutant BaxP168A was more active than BaxWT. Both Bax variants were then reconstituted in nanodiscs by co-formation. Size exclusion chromatography, dynamic light scattering and transmission electron microscopy showed that nanodiscs formed with BaxP168A were larger than nanodiscs formed with BaxWT. We calculated that nanodiscs containing BaxP168A displayed a pore having a diameter of about 4.8 nm. This was consistent with the hypothesis that BaxP168A was reconstituted in nanodiscs as an active oligomer, corresponding to an intermediate step between the initial insertion of active Bax monomers/dimers (forming a pore having a diameter of 3.5 nm) and the formation of large pores observed during late apoptosis (having a diameter above 40 nm).

The present study aimed to evaluate in vitro enzymatic activity of a novel biotechnological active complex based on natural origin compounds – thermophilic lipase and β-cyclodextrin – for hydrolysis of acylglycerides in plant oils and fats. β-cyclodextrin (β-CD) as an additive has attracted attention for enhance stability and efficiency of enzymes. In present study, the effects of β-CD on enzymatic hydrolysis of acylglycerides by thermophilic lipase were investigated by modern methods. The UV-spectroscopy, electron microscopy with TEM and kinetics of enzymatic hydrolysis were compared by the addition of β-CD, respectively. The results showed that lipase could produce the highest yield of oleic acid in presence of β-CD after 1 and 3h. The UV spectroscopy demonstrated that the absorbance and fluorescence of lipase decreased with increasing concentration of β-CD due to surface interaction and change of enzyme configuration. Moreover, electron microscopy with TEM showed that lipase formed a special active conglomerate with β-CD for improving hydrolysis and stability. Dermatology evaluation indicated that complex added to household products didn’t affect sensitive skin of hands. Overall results indicate that β-CD could increase enzymatic activity of the lipase against acylglycerides and can be considered as promising composition in ecological household products for regular hand application.

Since December 2019, the problem caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has grown to a global threat. The search for new treatment strategies is strongly associated with both fundamental research on mechanisms of virus life cycle and development of new screening platforms for anti-viral drug candidates. In this project we labelled SARS-CoV-2 membrane proteins M, E, S and studied their localization in mammalian cell compartments using fluorescent microscopy. We tested N- and C-oriented sensor designs and different fluorescent proteins. Also, we successfully visualized the early stages of M protein transport in real time. We found that M protein localizes in cell lysosomes, which supports the recent hypothesis that β-coronaviruses use lysosomal organelles for egress instead of traditional Golgi-mediated secretory pathway. Further we plan to study interactions between M, E and S using the FRET method. In addition, we developed several types of FRET-based and translocational sensors to track SARS-CoV-2 PLpro protease and measure its activity in living cells. Preliminary experiments showed the expected increase in donor fluorescence after proteolysis of PLpro site between the FRET-pair. Results of the current project will provide unique information on spatial-temporal dynamics and interaction between SARS-CoV-2 membrane proteins during the viral lifecycle. The developed system for real-time visualization of PLpro activity can potentially serve as a basis for safe cell antiviral drug screening platforms. The proposed strategy for studying viral proteins combines two important properties. Firstly, research is conducted on human living cells, which is closely approximated to native conditions in contrast to in vitro experiments. Secondly, the experimental system lacks interaction with a functional virus which makes it completely safe for the researcher.

Protein purification is an ever-vital approach for each academia and industry. This paper mainly reviews and discusses one of the center contents of proteomics, the latest advances in separation technology for protein components focusing on five different methods. The MCPA system is an economically amazing invention for protein therapy researchers to purify samples. Because of the excessive protease interest of Ulp1 toward SUMO fusion, excessive protein products may be done inside 1/2 of an hour through this method. The magnetic separation strategies will offer a higher method of protein purification withinside the close to imminent because of a few advantages. The evaluation established that the ATPS technique became a cost-effective, time-saving (30 min), and high-recuperation approach that can be scaled up for commercial purposes. Herein, ATPS may be an ability approach for the purification of single-step separation and keep away from multistep purification just like the chromatography technique. Therefore, our recent reviews provide a capable technique for competent protein purification.

Multidrug-resistant bacteria can lead to skin wound infections, pain and long-term treatment. The development of high-performance dressings for the treatment of infected skin wounds represents a necessary requirement. In this paper it was designed and synthesized adhesive hydrogel films containing active agents such as hyaluronic acid (HA) and lidocaine, as local anesthetic. HA is an extremely effective and long-lasting moisturizer, biocompatible, with a regenerating effect, promoting the regeneration of damaged skin. lidocaine has been incorporated to relieve acute pain and improve patient comfort and endurance. Films without HA and lidocaine were also produced as control sample. The successful synthesis of films obtained at room temperature, from aqueous solutions, was also confirmed by Fourier transform infrared analysis, X-ray diffraction and scanning electron analysis. Water absorption, adhesion and mechanical strength of the films-based hydrogel are improved with the introduction of HA and lidocaine, leading to the rapid skin wound healing process. The obtained materials will be used as promising materials able to restore the structural and functional properties of the skin. Wet adhesive backing films with good adhesion to skin, encapsulating HA with different molecular weights and lidocaine were developed to explore their potential to be used as a patch for painless treatment and healing of skin wounds. The cytocompatibility studies confirmed that the obtained films-based hydrogel have demonstrated no cytotoxic effect.

Cutaneous administration has advantages over the oral or intravenous route such as convenience for the patient, avoids hepatic metabolism, and provides patient, it avoids hepatic metabolism and provides sustained sustained administration of the active component over long periods of time. A major challenge in this route is the administration of drugs that are difficult to penetrate. For these, it is necessary to design delivery vehicles that help increase the stability of the active components and facilitate transport across the skin barrier. In this work, magnetoliposomes (MLPs) immobilizing magnetite nanoparticles (MNPs) have been realized. MNPs act as a nanocarrier for hydrophobic drugs, such as doxorubicin (DOX). To facilitate topical application, MLPs are dispersed in photoresponsive methacrylated chitosan hydrogels. For this purpose, the MLPs were synthesized by coprecipitation of FeCl3 and FeCl2. Subsequently, they were silanized and functionalized a PEG spacer to bind DOX. The success of each functionalization step was evaluated by Fourier transform infrared spectroscopy (FTIR) and thermogravimetric analysis (TGA). The size and morphology of the PEG-DOX-MNPs were analyzed by DLS and TEM. Then, the MNPs-PEG-DOX MNPs were encapsulated in liposomes synthesized by layer hydration method. Dispersion of MLPs in the hydrogel followed by crosslinking with visible blue light was performed. Preliminary FTIR results indicate a correct synthesis and functionalization of the MNPs, as indicated by the presence of bands corresponding to the Si-O stretching vibration at 1029 cm-1, Fe-O absorption bands around 560 cm-1. TGA results showed a weight loss of 3.5% for MNPs from 200 to 400°C, which was attributed to silane ligands. The hydrodynamic diameter of the MNPs was 140 nm with polydispersity indices of 0.16. In future work, DOX will be conjugated to MNPs and MLPs will be synthesized for dispersion in the hydrogel. Subsequently, drug release kinetics tests will be performed under relevant conditions.

Gene therapy is a relatively new discipline of molecular medicine that will have a long-term impact on human health [1]. Its goal is to treat diseases through genome editing and gene expression modification. Gene therapy approaches are used to treat a variety of disorders, including neurological and infectious diseases, as well as monogenic and eye diseases [2–5]. Viral vectors, antisense oligonucleotides (ASO), RNA interference (RNAi), plasmids, deoxyribozymes (DZs), and CRISPR/Cas9 are some of the gene therapies under development [6-7]. However, studies have shown that such approaches have off-target effects, low affinity to folded RNA and are expensive (CRISPR and RNAi) (citation needed). Modern methods of addressing such issues include chemical modification of molecular tools and computer design. But such approaches are still being studied and do not guarantee high therapeutic effects [8]. DNAzyme, or deoxyribozyme (DZ), are single-stranded DNA molecular catalysts obtained through in-vitro screening technology [10] and can catalyze a variety of reactions, including RNA and DNA cleavage and ligation, as well as DNA phosphorylation [11]. For ion-dependent catalysis, they do not recruit enzymes in comparison with ASO and RNAi and Dzs are also simple in design, which makes them promising tools for RNA-cleaving gene therapy. In this work, to increase affinity for target RNA-cleaving DNAzymes, we developed bivalent DNAzymes (BDs), made of two Dz-ligands capable of RNA2 cleaving in several sites (Fig. 1). As in nature, the concept of multivalent DNAzyme consists of multiple ligands that bind to their target at multiple sites with high affinity and avidity resulting in effective inhibition or stimulation of biological responses [12]. Scientists have reported multivalency in biological systems as a powerful strategy for achieving high-affinity molecular recognition [13]. As original DNAzymes, BDs allow inhibition of expression of critical targeted genes via catalytic cleavage of mRNA, but their effectiveness is determined by avidity (which can be considered as the sum of the affinity of each active ligand containing the drug) instead of affinity, and we suppose that this parameter will increase drug-target interaction. We hope that such an approach can offer a promising future for improving gene therapy.

Our main goal was to improve the affinity and efficiency of DNAzymecleaving RNA by developing and optimizing DZ 10-23 base gene therapy molecules. We designed BDs that target folded mRNA and tested their efficiency in an in vitro physiological buffer to establish conditions necessary for effective gene knockdown. Next, we optimized DZ 10-23 by designing monovalent DZs with varied arm lengths (short arms with 1-2 nucleotides less and long arms with1-2 nucleotides more) to find the most efficient and stable construct. Finally, a larger RNA (STR-104) study was conducted to demonstrate the constructs' efficiency and stability with a larger or different target. BDs demonstrated higher cleavage efficiency (63.5%) in comparison with monovalent DZs and DZ associations of DZ1-DZ2 (9.2% and 23.4%, respectively). The most efficient multivalent design and the DZ1-DZ2 association were used to test the efficiency of both multivalent and monovalent designs at different concentrations (10nM, 25nM, 50nM, and 100nM). BDs at low concentration (25nM) was more efficient (33%) than DZ1-DZ2 (21%) at a higher concentration (100nM). Further investigation with different lengths (-/+1, -/+2 nucleotides) of binding arms and different melting temperatures of the linker tails influenced the efficiency of the designs. The result indicated that, as the length of the binding arms and the melting temperature of the designs increase, the efficiency also increases. This research has demonstrated that multivalent DNAzymes have great potential to increase DNAzyme-cleaving RNA affinity and efficiency as a therapeutic agent.

References

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