L-Asparaginase Conjugates from the Hyperthermophilic Archaea Thermococcus sibiricus with Improved Biocatalytic Properties.

This study investigated the effect of polycationic and uncharged polymers (and oligomers) on the catalytic parameters and thermostability of L-asparaginase from Thermococcus sibiricus (TsA). This enzyme has potential applications in the food industry to decrease the formation of carcinogenic acrylamide during the processing of carbohydrate-containing products. Conjugation with the polyamines polyethylenimine and spermine (PEI and Spm) or polyethylene glycol (PEG) did not significantly affect the secondary structure of the enzyme. PEG contributes to the stabilization of the dimeric form of TsA, as shown by HPLC. Furthermore, neither polyamines nor PEG significantly affected the binding of the L-Asn substrate to TsA. The conjugates showed greater maximum activity at pH 7.5 and 85 °C, 10-50% more than for native TsA. The pH optima for both TsA-PEI and TsA-Spm conjugates were shifted to lower pH ranges from pH 10 (for the native enzyme) to pH 8.0. Additionally, the TsA-Spm conjugate exhibited the highest activity at pH 6.5-9.0 among all the samples. Furthermore, the temperature optimum for activity at pH 7.5 shifted from 90-95 °C to 80-85 °C for the conjugates. The thermal inactivation mechanism of TsA-PEG appeared to change, and no aggregation was observed in contrast to that of the native enzyme. This was visually confirmed and supported by the analysis of the CD spectra, which remained almost unchanged after heating the conjugate solution. These results suggest that TsA-PEG may be a more stable form of TsA, making it a potentially more suitable option for industrial use.

Engineering and Expression Strategies for Optimization of L-Asparaginase Development and Production.

Genetic engineering for heterologous expression has advanced in recent years. Model systems such as Escherichia coli, Bacillus subtilis and Pichia pastoris are often used as host microorganisms for the enzymatic production of L-asparaginase, an enzyme widely used in the clinic for the treatment of leukemia and in bakeries for the reduction of acrylamide. Newly developed recombinant L-asparaginase (L-ASNase) may have a low affinity for asparagine, reduced catalytic activity, low stability, and increased glutaminase activity or immunogenicity. Some successful commercial preparations of L-ASNase are now available. Therefore, obtaining novel L-ASNases with improved properties suitable for food or clinical applications remains a challenge. The combination of rational design and/or directed evolution and heterologous expression has been used to create enzymes with desired characteristics. Computer design, combined with other methods, could make it possible to generate mutant libraries of novel L-ASNases without costly and time-consuming efforts. In this review, we summarize the strategies and approaches for obtaining and developing L-ASNase with improved properties.

Cytoprotective Activity of Polyamines Is Associated with the Alternative Splicing of RAD51A Pre-mRNA in Normal Human CD4+ T Lymphocytes.

Physiological polyamines are ubiquitous polycations with pleiotropic biochemical activities, including regulation of gene expression and cell proliferation as well as modulation of cell signaling. They can also decrease DNA damage and promote cell survival. In the present study, we demonstrated that polyamines have cytoprotective effects on normal human CD4+ T lymphocytes but not on cancer Jurkat or K562 cells. Pretreatment of lymphocytes with polyamines resulted in a significant reduction in cells with DNA damage induced by doxorubicin, cisplatin, or irinotecan, leading to an increase in cell survival and viability. The induction of RAD51A expression was in response to DNA damage in both cancer and normal cells. However, in normal cells, putrescin pretreatment resulted in alternative splicing of RAD51A and the switch of the predominant expression from the splice variant with the deletion of exon 4 to the full-length variant. Induction of RAD51A alternative splicing by splice-switching oligonucleotides resulted in a decrease in DNA damage and cell protection against cisplatin-induced apoptosis. The results of this study suggest that the cytoprotective activity of polyamines is associated with the alternative splicing of RAD51A pre-mRNA in normal human CD4+ T lymphocytes. The difference in the sensitivity of normal and cancer cells to polyamines may become the basis for the use of these compounds to protect normal lymphocytes during lymphoblastic chemotherapy.

Anticancer Cytotoxic Activity of Bispidine Derivatives Associated with the Increasing Catabolism of Polyamines.

Polyamine (PA) catabolism is often reduced in cancer cells. The activation of this metabolic pathway produces cytotoxic substances that might cause apoptosis in cancer cells. Chemical compounds able to restore the level of PA catabolism in tumors could become potential antineoplastic agents. The search for activators of PA catabolism among bicyclononan-9-ones is a promising strategy for drug development. The aim of the study was to evaluate the biological activity of new 3,7-diazabicyclo[3.3.1]nonan-9-one derivatives that have antiproliferative properties by accelerating PA catabolism. Eight bispidine derivatives were synthetized and demonstrated the ability to activate PA catabolism in regenerating rat liver homogenates. However, only three of them demonstrated a potent ability to decrease the viability of cancer cells in the MTT assay. Compounds 4c and 4e could induce apoptosis more effectively in cancer HepG2 cells rather than in normal WI-38 fibroblasts. The lead compound 4e could significantly enhance cancer cell death, but not the death of normal cells if PAs were added to the cell culture media. Thus, the bispidine derivative 4e 3-(3-methoxypropyl)-7-[3-(1H-piperazin-1-yl)ethyl]-3,7-diazabicyclo[3.3.1]nonane could become a potential anticancer drug substance whose mechanism relies on the induction of PA catabolism in cancer cells.

Tissue-Specific Expression of Genes Involved in Cellular Transportation in Common Carp (Cyprinus carpio) Exposed to Cadmium.

Juvenile common carp were treated with Cd2+ at a sublethal concentration for Cyprinidae (6.4 mg/L). The expression of N-methyl-D-aspartate receptor subunit genes (NR2A, NR2B) and ATP-binding cassette subfamily C member 1 gene (ABCC1) was compared between treated and untreated fish. In addition, cadmium accumulation in the fish tissues was assessed. NR2A was 18.9-fold upregulated by Cd2+ in the eyes (choroid + retina), which accumulated Cd, and was not upregulated in brain, which didn't accumulate Cd. This may have been caused by the blocking of calcium channels by Cd2+, which has a very similar ionic radius to that of Ca2+. ABCC1 was 2.6-fold upregulated in gills and was not upregulated in liver; both tissues accumulated high levels of Cd. This difference may have been caused by the accumulation of predominantly previously inactivated Cd in liver or by some difference in the mechanisms of self-detoxification from Cd2+ in fish gills and liver.

DNase I Induces Other Endonucleases in Kidney Tubular Epithelial Cells by Its DNA-Degrading Activity.

Endonuclease-mediated DNA fragmentation is both an immediate cause and a result of apoptosis and of all other types of irreversible cell death after injury. It is produced by nine enzymes including DNase I, DNase 2, their homologs, caspase-activated DNase (CAD) and endonuclease G (EndoG). The endonucleases act simultaneously during cell death; however, regulatory links between these enzymes have not been established. We hypothesized that DNase I, the most abundant of endonucleases, may regulate other endonucleases. To test this hypothesis, rat kidney tubular epithelial NRK-52E cells were transfected with the DNase I gene or its inactive mutant in a pECFP expression vector, while control cells were transfected with the empty vector. mRNA expression of all nine endonucleases was studied using real-time RT-PCR; DNA strand breaks in endonuclease genes were determined by PCR and protein expression of the enzymes was measured by Western blotting and quantitative immunocytochemistry. Our data showed that DNase I, but not its inactive mutant, induces all other endonucleases at varying time periods after transfection, causes DNA breaks in endonuclease genes, and elevates protein expression of several endonucleases. This is the first evidence that endonucleases seem to be induced by the DNA-degrading activity of DNase I.

Inhibition of telomerase activity by splice-switching oligonucleotides targeting the mRNA of the telomerase catalytic subunit affects proliferation of human CD4+ T lymphocytes.

Telomerase activity is regulated at the mRNA level by alternative splicing (AS) of its catalytic subunit hTERT. The aim of this study was to define the ability of splice-switching oligonucleotides (SSOs) that pair with hTERT pre-mRNA to induce AS and inhibit telomerase activity in human CD4+ T lymphocytes. SSOs that blocked the binding of a single splicing regulatory protein, SRp20 or SRp40, to its site within intron 8 of hTERT pre-mRNA demonstrated rather moderate capacities to induce AS and inhibit telomerase. However, a SSO that blocked the interaction of both SRp20 and SRp40 proteins with pre-mRNA was the most active. Cultivation of lymphocytes with spliced hTERT and inhibited telomerase resulted in the reduction of proliferative activity without significant induction of cell death. These results should facilitate further investigation of telomerase activity regulation, and antitelomerase SSOs could become promising agents for antiproliferative cell therapy.

Murine regulatory T cells induce death of effector T, B, and NK lymphocytes through a contact-independent mechanism involving telomerase suppression and telomere-associated senescence.

Regulatory T cells (Tregs) suppress the activity of effector T, B and NK lymphocytes and sustain immunological tolerance, but the proliferative activity of suppressed cells remains unexplored. In the present study, we report that mouse Tregs can induce replicative senescence and the death of responder mouse CD4+CD25- T cells, CD8+ T cells, B cells and NK cells in vitro and in vivo. Contact-independent in vitro co-cultivation with Tregs up-regulated endonuclease G (EndoG) expression and its translocation to the nucleus in responder cells. EndoG localization in the nucleus induced alternative mRNA splicing of the telomerase catalytic subunit Tert and telomerase inhibition. The lack of telomerase activity in proliferating cells led to telomere loss followed by the development of senescence and cell death. Injection of Tregs into mice resulted in EndoG-associated alternative splicing of Tert, telomerase inhibition, telomere loss, senescence development and increased cell death in vivo. The present study describes a novel contact-independent mechanism by which Tregs specify effector cell fate and provides new insights into cellular crosstalk related to immune suppression.

Rhodospirillum rubruml-asparaginase targets tumor growth by a dual mechanism involving telomerase inhibition.

Rhodospirillum rubruml-asparaginase mutant RrA E149R, V150P, F151T (RrA) was previously identified to down-regulate telomerase activity along with catalyzing the hydrolysis of l-asparagine. The aim of this study was to define the effect of prolonged RrA exposure on telomerase activity, maintenance of telomeres and proliferation of cancer cells in vitro and in vivo. RrA could inhibit telomerase activity in SCOV-3, SkBr-3 and A549 human cancer cell lines due to its ability to down-regulate the expression of telomerase catalytic subunit hTERT. Telomerase activity in treated cells did not exceeded 29.63 ± 12.3% of control cells. Continuous RrA exposure of these cells resulted in shortening of telomeres followed by cell death in vitro. Using real time PCR we showed that length of telomeres in SCOV-3 cells has been gradually decreasing from 10105 ± 2530 b.p. to 1233 ± 636 b.p. after 35 days of cultivation. RrA treatment of xenograft models in vivo showed slight inhibition of tumor growth accompanied with 49.5-53.3% of decrease in hTERT expression in the all tumors. However down-regulation of hTERT expression, inhibition of telomerase activity and the loss of telomeres was significant in response to RrA administration in xenograft models. These results should facilitate further investigations of RrA as a potent therapeutic protein.

Many Faces of Regulatory T Cells: Heterogeneity or Plasticity?

Regulatory T cells (Tregs) are essential for maintaining the immune balance in normal and pathological conditions. In autoimmune diseases and transplantation, they restrain the loss of self-tolerance and promote engraftment, whereas in cancer, an increase in Treg numbers is mostly associated with tumor growth and poor prognosis. Numerous markers and their combinations have been used to identify Treg subsets, demonstrating the phenotypic diversity of Tregs. The complexity of Treg identification can be hampered by the unstable expression of some markers, the decrease in the expression of a specific marker over time or the emergence of a new marker. It remains unclear whether such phenotypic shifts are due to new conditions or whether the observed changes are due to initially different populations. In the first case, cellular plasticity is observed, whereas in the second, cellular heterogeneity is observed. The difference between these terms in relation to Tregs is rather blurred. Considering the promising perspectives of Tregs in regenerative cell-based therapy, the existing confusing data on Treg phenotypes require further investigation and analysis. In our review, we introduce criteria that allow us to distinguish between the heterogeneity and plasticity of Tregs normally and pathologically, taking a closer look at their diversity and drawing the line between two terms.

Electrochemical approach for the analysis of DNA degradation in native DNA and apoptotic cells.

The aim of this work was to develop an electrochemical approach for the analysis of DNA degradation and fragmentation in apoptotic cells. DNA damage is considered one of the major causes of human diseases. We analyzed the cleavage processes of the circular plasmid pTagGFP2-N and calf thymus DNA, which were exposed to restriction endonucleases (the restriction endonucleases BstMC I and AluB I and the nonspecific endonuclease I). Genomic DNA from the leukemia K562 cell line was used as a marker of the early and late (mature) stages of apoptosis. Registration of direct electrochemical oxidation of nucleobases of DNA molecules subjected to restriction endonuclease or apoptosis processes was proposed for the detection of these biochemical events. Label-free differential pulse voltammetry (DPV) has been used to measure endonuclease activities and DNA damage using carbon nanotube-modified electrodes. The present DPV technique provides a promising platform for high-throughput screening of DNA hydrolases and for registering the efficiency of apoptotic processes. DPV comparative analysis of the circular plasmid pTagGFP2-N in its native supercoiled state and plasmids restricted to 4 and 23 parts revealed significant differences in their electrochemical behavior. Electrochemical analysis was fully confirmed by means of traditional methods of DNA analysis and registration of apoptotic process, such as gel electrophoresis and flow cytometry.

Correction: Plyasova et al. Penetration into Cancer Cells via Clathrin-Dependent Mechanism Allows L-Asparaginase from Rhodospirillum rubrum to Inhibit Telomerase. Pharmaceuticals 2020, 13, 286.

In the original publication [...].

Induction of FoxP3 Pre-mRNA Alternative Splicing to Enhance the Suppressive Activity of Regulatory T Cells from Amyotrophic Lateral Sclerosis Patients.

Forkhead box protein 3 (FoxP3) is a key transcription factor responsible for the development, maturation, and function of regulatory T cells (Tregs). The FoxP3 pre-mRNA is subject to alternative splicing, resulting in the translation of multiple splice variants. We have shown that Tregs from patients with amyotrophic lateral sclerosis (ALS) have reduced expression of full-length (FL) FoxP3, while other truncated splice variants are expressed predominantly. A correlation was observed between the reduced number of Tregs in the peripheral blood of ALS patients, reduced total FoxP3 mRNA, and reduced mRNA of its FL splice variant. Induction of FL FoxP3 was achieved using splice-switching oligonucleotides capable of base pairing with FoxP3 pre-mRNA and selectively modulating the inclusion of exons 2 and 7 in the mature mRNA. Selective expression of FL FoxP3 resulted in the induction of CD127low, CD152, and Helios-positive cells, while the cell markers CD4 and CD25 were not altered. Such Tregs had an increased proliferative activity and a higher frequency of cell divisions per day. The increased suppressive activity of Tregs with the induced FL FoxP3 splice variant was associated with the increased synthesis of the pro-apoptotic granzymes A and B, and perforin, IL-10, and IL-35, which are responsible for contact-independent suppression, and with the increased ability to suppress telomerase in target cells. The upregulation of Treg suppressive and proliferative activity using splice-switching oligonucleotides to induce the predominant expression of the FoxP3 FL variant is a promising approach for regenerative cell therapy in Treg-associated diseases.

DNA-Based Nanomaterials as Drug Delivery Platforms for Increasing the Effect of Drugs in Tumors.

DNA nanotechnology has significantly advanced and might be used in biomedical applications, drug delivery, and cancer treatment during the past few decades. DNA nanomaterials are widely used in biomedical research involving biosensing, bioimaging, and drug delivery since they are remarkably addressable and biocompatible. Gradually, modified nucleic acids have begun to be employed to construct multifunctional DNA nanostructures with a variety of architectural designs. Aptamers are single-stranded nucleic acids (both DNAs and RNAs) capable of self-pairing to acquire secondary structure and of specifically binding with the target. Diagnosis and tumor therapy are prospective fields in which aptamers can be applied. Many DNA nanomaterials with three-dimensional structures have been studied as drug delivery systems for different anticancer medications or gene therapy agents. Different chemical alterations can be employed to construct a wide range of modified DNA nanostructures. Chemically altered DNA-based nanomaterials are useful for drug delivery because of their improved stability and inclusion of functional groups. In this work, the most common oligonucleotide nanomaterials were reviewed as modern drug delivery systems in tumor cells.

The Role of Regulatory T Cells in the Onset and Progression of Primary Sjögren's Syndrome.

Regulatory T cells (Tregs) play a key role in maintaining immune balance and regulating the loss of self-tolerance mechanisms in various autoimmune diseases, including primary Sjögren's syndrome (pSS). With the development of pSS primarily in the exocrine glands, lymphocytic infiltration occurs in the early stages, mainly due to activated CD4+ T cells. Subsequently, in the absence of rational therapy, patients develop ectopic lymphoid structures and lymphomas. While the suppression of autoactivated CD4+ T cells is involved in the pathological process, the main role belongs to Tregs, making them a target for research and possible regenerative therapy. However, the available information about their role in the onset and progression of this disease seems unsystematized and, in certain aspects, controversial. In our review, we aimed to organize the data on the role of Tregs in the pathogenesis of pSS, as well as to discuss possible strategies of cell therapy for this disease. This review provides information on the differentiation, activation, and suppressive functions of Tregs and the role of the FoxP3 protein in these processes. It also highlights data on various subpopulations of Tregs in pSS, their proportion in the peripheral blood and minor salivary glands of patients as well as their role in the development of ectopic lymphoid structures. Our data emphasize the need for further research on Tregs and highlight their potential use as a cell-based therapy.

Oxazolinyl derivatives of androst-16-ene as inhibitors of CYP17A1 activity and prostate carcinoma cells proliferation: Effects of substituents in oxazolinyl moiety.

Steroid derivatives modified with nitrogen containing heterocycles are known to inhibit activity of steroidogenic enzymes, decrease proliferation of cancer cells and attract attention as promising anticancer agents. Specifically, 2'-(3ß-hydroxyandrosta-5,16-dien-17-yl)-4',5'-dihydro-1',3'-oxazole 1a potently inhibited proliferation of prostate carcinoma cells. In this study we synthesized and investigated five new derivatives of 3ß-hydroxyandrosta-5,16-diene comprising 4'-methyl or 4'-phenyl substituted oxazolinyl cycle 1 (b-f). Docking of compounds 1 (a-f) to CYP17A1 active site revealed that the presence of substitutents at C4' atom in oxazoline cycle, as well as C4' atom configuration, significantly affect docking poses of compounds in the complexes with enzyme. Testing of compounds 1 (a-f) as CYP17A1 inhibitors revealed that the only compound 1a, comprising unsubstituted oxazolinyl moiety, demonstrated strong inhibitory activity, while other compounds 1 (b-f) were slightly active or non active. Compounds 1 (a-f) efficiently decreased growth and proliferation of prostate carcinoma LNCaP and PC-3 cells at 96 h incubation; the effect of compound 1a was the most powerful. Compound 1a efficiently stimulated apoptosis and caused PC-3 cells death, that was demonstrated by a direct comparison of pro-apoptotic effects of compound 1a and abiraterone.

Effect of the Teeth Whitening Procedure on the Mineral Composition of Oral Fluid.

The basis of modern tooth whitening systems is the use of a whitening gel, which usually contains hydrogen peroxide or carbamide peroxide. The study included 81 patients aged 22 to 35 years with a tooth color A2 and a darker color on the Vita Classic scale. The purpose of our research was to identify a new approach to whitening teeth to improve safety and gentleness. To perform this, we assessed the effect of the tooth whitening procedure on the mineral composition of the oral fluid. A new approach to the teeth whitening procedure was to use a mouth retractor and a tool for aspirating the whitening gel, which we developed. Before the procedure, a protective film-forming aerosol, which included sodium ascorbate, was applied. After the tooth whitening procedure, the enamel was remineralized with a sealing liquid for 14 days. The concentrations of calcium and phosphorus in the oral fluid were determined using a spectrophotometer with a set of reagents (Human). The results obtained indicate that the new approach to the teeth whitening procedure contributed to less pronounced changes in the concentrations of calcium (+29.07, p < 0.001) and phosphorus (-14%, p < 0.001) in the oral fluid immediately after the procedure and in combination with the standard procedure for teeth whitening; immediately after this procedure, the calcium concentration increased by 74.4% (p < 0.001), and the phosphorus concentration decreased by 23.07% (p < 0.001). The use of remineralizing agents led to a faster recovery of the initial levels of calcium and phosphorus in the oral fluid.

Modulation of Suppressive Activity and Proliferation of Human Regulatory T Cells by Splice-Switching Oligonucleotides Targeting FoxP3 Pre-mRNA.

The maturation, development, and function of regulatory T cells (Tregs) are under the control of the crucial transcription factor Forkhead Box Protein 3 (FoxP3). Through alternative splicing, the human FoxP3 gene produces four different splice variants: a full-length variant (FL) and truncated variants with deletions of each of exons 2 (∆2 variant) or 7 (∆7 variant) or a deletion of both exons (∆2∆7 variant). Their involvement in the biology of Tregs as well as their association with autoimmune diseases remains to be clarified. The aim of this work was to induce a single FoxP3 splice variant in human Tregs by splice switching oligonucleotides and to monitor their phenotype and proliferative and suppressive activity. We demonstrated that Tregs from peripheral blood from patients with multiple sclerosis preferentially expressed truncated splice variants, while the FL variant was the major variant in healthy donors. Tregs with induced expression of truncated FoxP3 splice variants demonstrated lower suppressive activity than those expressing FL variants. Reduced suppression was associated with the decreased expression of Treg-associated suppressive surface molecules and the production of cytokines. The deletion of exons 2 and/or 7 also reduced the cell proliferation rate. The results of this study show an association between FoxP3 splice variants and Treg function and proliferation. The modulation of Treg suppressive activity by the induction of the FoxP3 FL variant can become a promising strategy for regenerative immunotherapy.

Molecular Analysis of L-Asparaginases for Clarification of the Mechanism of Action and Optimization of Pharmacological Functions.

L-asparaginases (EC 3.5.1.1) are a family of enzymes that catalyze the hydrolysis of L-asparagine to L-aspartic acid and ammonia. These proteins with different biochemical, physicochemical and pharmacological properties are found in many organisms, including bacteria, fungi, algae, plants and mammals. To date, asparaginases from E. coli and Dickeya dadantii (formerly known as Erwinia chrysanthemi) are widely used in hematology for the treatment of lymphoblastic leukemias. However, their medical use is limited by side effects associated with the ability of these enzymes to hydrolyze L-glutamine, as well as the development of immune reactions. To solve these issues, gene-editing methods to introduce amino-acid substitutions of the enzyme are implemented. In this review, we focused on molecular analysis of the mechanism of enzyme action and to optimize the antitumor activity.

Improvement of Biocatalytic Properties and Cytotoxic Activity of L-Asparaginase from Rhodospirillum rubrum by Conjugation with Chitosan-Based Cationic Polyelectrolytes.

L-asparaginases (L-ASNases, EC 3.5.1.1) are a family of enzymes that are widely used for the treatment of lymphoblastic leukemias. L-ASNase from Rhodospirillum rubrum (RrA) has a low molecular weight, low glutaminase activity, and low immunogenicity, making it a promising enzyme for antitumor drug development. In our work, the complex formation and covalent conjugation of the enzyme with synthetic or natural polycationic polymers was studied. Among non-covalent polyelectrolyte complexes (PEC), polyethyleneimine (PEI) yielded the highest effect on RrA, increasing its activity by 30%. The RrA-PEI complex had increased stability to trypsinolysis, with an inactivation constant decrease up to 10-fold compared to that of the native enzyme. The covalent conjugation of RrA with chitosan-PEI, chitosan-polyethylene glycol (chitosan-PEG), and chitosan-glycol resulted in an increase in the specific activity of L-asparagine (up to 30%). RrA-chitosan-PEG demonstrated dramatically (by 60%) increased cytotoxic activity for human chronic myeloma leukemia K562 cells in comparison to the native enzyme. The antiproliferative activity of RrA and its conjugates was significantly higher (up to 50%) than for that of the commercially available EcA at the same concentration. The results of this study demonstrated that RrA conjugates with polycations can become a promising strategy for antitumor drug development.