Non-viral-mediated gene transfer of OX40 ligand for tumor immunotherapy

BackgroundImmune checkpoint blockade (ICB) is rapidly becoming a standard of care in the treatment of many cancer types. However, the subset of patients who respond to this type of therapy is limited. Another way to promote antitumoral immunity is the use of immunostimulatory molecules, such as cytokines or T cell co-stimulators. The systemic administration of immunotherapeutics leads to significant immune-related adverse events (irAEs), therefore, the localized antitumoral action is needed. One way to achieve this is intratumoral non-viral gene-immune therapy, which allows for prolonged and localized gene expression, and multiple drug administration. In this study, we combined the previously described non-viral gene delivery system, PEG-PEI-TAT copolymer, PPT, with murine OX40L-encoding plasmid DNA.MethodsThe resulting OX40L/PPT nanoparticles were characterized via gel mobility assay, dynamic light scattering analysis and in vitro transfection efficiency evaluation. The antitumoral efficacy of intratumorally (i.t.) administered nanoparticles was estimated using subcutaneously (s.c.) implanted CT26 (colon cancer), B16F0 (melanoma) and 4T1 (breast cancer) tumor models. The dynamics of stromal immune cell populations was analyzed using flow cytometry. Weight loss and cachexia were used as irAE indicators. The effect of combination of i.t. OX40L/PPT with intraperitoneal PD-1 ICB was estimated in s.c. CT26 tumor model.ResultsThe obtained OX40L/PPT nanoparticles had properties applicable for cell transfection and provided OX40L protein expression in vitro in all three investigated cancer models. We observed that OX40L/PPT treatment successfully inhibited tumor growth in B16F0 and CT26 tumor models and showed a tendency to inhibit 4T1 tumor growth. In B16F0 tumor model, OX40L/PPT treatment led to the increase in antitumoral effector NK and T killer cells and to the decrease in pro-tumoral myeloid cells populations within tumor stroma. No irAE signs were observed in all 3 tumor models, which indicates good treatment tolerability in mice. Combining OX40L/PPT with PD-1 ICB significantly improved treatment efficacy in the CT26 subcutaneous colon cancer model, providing protective immunity against CT26 colon cancer cells.ConclusionOverall, the anti-tumor efficacy observed with OX40L non-viral gene therapy, whether administered alone or in combination with ICB, highlights its potential to revolutionize cancer gene therapy, thus paving the way for unprecedented advancements in the cancer therapy field

THE SEMANTIC STRUCTURE OF LANDSCAPE TERMS IN GERMAN FOLK TEXTS (A CASE STUDY OF FAIRY TALES, PROVERBS AND SAYINGS)

German folk landscape lexis, its functioning and text components' influence are studied in the aspect of interaction of some factors, such as a key role of denotative components while conceptualizing landscape's objects and their material characteristics; folk genre's purpose while actualizing its connotative features of social space; the influence of text units (actualizators) and their semantics on 'expansion' of meanings in landscape term structures. Due to the analysis of functional features of these words, two main features of the space have been determined – representation of landscape's objects and their material characteristics (size, length, quantity, material, stuff) and realization of their social features ('safe/unsafe', 'unavailable/available', 'useless/useful', etc.). The dominance of the material component in the semantic structure of landscape terms is noted in the designations of mountainous terrain. The social characteristics are presented with subjective interpretation of individual properties and elements of the relief. The conditions of interaction between the members and the landscape contexts are disclosed in the analysis of phrases in which the social characteristics of the complexes express meanings ('size', 'unavailable', 'safe', etc.). The study shows that folk texts of different genres (fairy tales, proverbs and sayings) is a valuable source of lexical material, as they reflect the stable interactions between connotative components and the semantics of fairy tales texts, proverbs and sayings. Thus, they should be taken into consideration while translating German folk texts to Russian language

Myelin Basic Protein Attenuates Furin-Mediated Bri2 Cleavage and Postpones Its Membrane Trafficking

Myelin basic protein (MBP) is the second most abundant protein in the central nervous system and is responsible for structural maintenance of the myelin sheath covering axons. Previously, we showed that MBP has a more proactive role in the oligodendrocyte homeostasis, interacting with membrane-associated proteins, including integral membrane protein 2B (ITM2B or Bri2) that is associated with familial dementias. Here, we report that the molecular dynamics of the in silico-generated MBP-Bri2 complex revealed that MBP covers a significant portion of the Bri2 ectodomain, assumingly trapping the furin cleavage site, while the surface of the BRICHOS domain, which is responsible for the multimerization and activation of the Bri2 high-molecular-weight oligomer chaperone function, remains unmasked. These observations were supported by the co-expression of MBP with Bri2, its mature form, and disease-associated mutants, which showed that in mammalian cells, MBP indeed modulates the post-translational processing of Bri2 by restriction of the furin-catalyzed release of its C-terminal peptide. Moreover, we showed that the co-expression of MBP and Bri2 also leads to an altered cellular localization of Bri2, restricting its membrane trafficking independently of the MBP-mediated suppression of the Bri2 C-terminal peptide release. Further investigations should elucidate if these observations have physiological meaning in terms of Bri2 as a MBP chaperone activated by the MBP-dependent postponement of Bri2 membrane trafficking

Probing the Role of a Conserved Phenylalanine in the Active Site of Thiocyanate Dehydrogenase

Copper-containing enzymes catalyze a broad spectrum of redox reactions. Thiocyanate dehydrogenase (TcDH) from Thioalkalivibrio paradoxus Arh1 enables the bacterium to use thiocyanate as a unique source of energy and nitrogen. Oxidation of thiocyanate takes place in the trinuclear copper center of TcDH with peculiar organization. Despite the TcDH crystal structure being established, a role of some residues in the enzyme active site has yet to be obscured. F436 residue is located in the enzyme active site and conserved among a number of TcDH homologs, however, its role in the copper center formation or the catalytic process is still not clear. To address this question, a mutant form of the enzyme with F436Q substitution (TcDHF436Q) was obtained, biochemically characterized, and its crystal structure was determined. The TcDHF436Q had an unaltered protein fold but did not possess enzymatic activity, whereas it contained all three copper ions, according to ICP-MS data. The structural data showed that the F436Q substitution resulted in a disturbance of hydrophobic interactions within the active site crucial for a correct transition between open/closed forms of the enzyme–substrate channel. Thus, we demonstrated that F436 does not participate in copper ion binding, but rather possesses a structural role in the TcDH active site

Probing the Role of a Conserved Phenylalanine in the Active Site of Thiocyanate Dehydrogenase

Copper-containing enzymes catalyze a broad spectrum of redox reactions. Thiocyanate dehydrogenase (TcDH) from Thioalkalivibrio paradoxus Arh1 enables the bacterium to use thiocyanate as a unique source of energy and nitrogen. Oxidation of thiocyanate takes place in the trinuclear copper center of TcDH with peculiar organization. Despite the TcDH crystal structure being established, a role of some residues in the enzyme active site has yet to be obscured. F436 residue is located in the enzyme active site and conserved among a number of TcDH homologs, however, its role in the copper center formation or the catalytic process is still not clear. To address this question, a mutant form of the enzyme with F436Q substitution (TcDHF436Q) was obtained, biochemically characterized, and its crystal structure was determined. The TcDHF436Q had an unaltered protein fold but did not possess enzymatic activity, whereas it contained all three copper ions, according to ICP-MS data. The structural data showed that the F436Q substitution resulted in a disturbance of hydrophobic interactions within the active site crucial for a correct transition between open/closed forms of the enzyme–substrate channel. Thus, we demonstrated that F436 does not participate in copper ion binding, but rather possesses a structural role in the TcDH active site

Proteomic dataset: Profiling of cultivated Echerichia coli isolates from Crohn's disease patients and healthy individuals

One of the dysbioses often observed in Crohn's disease (CD) patients is an increased abundance of Escherichia coli (10–100 fold compared to healthy individuals) (Gevers et al., 2014). The data reported is a large-scale proteome profile for E. coli isolates collected from CD patients and healthy individuals. 43 isolates were achieved from 30 CD patients (17 male, 12 female, median age 30) and 19 isolates from 7 healthy individuals (7 male, median age 19). Isolates were cultivated on LB medium at aerobic conditions up to medium log phase. Protein extraction was performed with sodium deoxycholate (DCNa) and urea, alcylation with tris(2-carboxyethyl)phosphine and iodacetamide. Protein trypsinolysis was performed as described in (Matyushkina et al., 2016). Total cell proteomes were analysed by shotgun proteomics with HPLC-MS/MS on a maXis qTOF mass-spectrometer. The data including HPLC-MS/MS raw files and exported Mascot search results was deposited to the PRIDE repository project accession: PXD010920, project https://doi.org/10.6019/PXD010920. Keywords: E. coli, Proteome, Crohn's disease, HPLC-MS/M

Unusual Cytochrome c552 from Thioalkalivibrio paradoxus: Solution NMR Structure and Interaction with Thiocyanate Dehydrogenase

The search of a putative physiological electron acceptor for thiocyanate dehydrogenase (TcDH) newly discovered in the thiocyanate-oxidizing bacteria Thioalkalivibrio paradoxus revealed an unusually large, single-heme cytochrome c (CytC552), which was co-purified with TcDH from the periplasm. Recombinant CytC552, produced in Escherichia coli as a mature protein without a signal peptide, has spectral properties similar to the endogenous protein and serves as an in vitro electron acceptor in the TcDH-catalyzed reaction. The CytC552 structure determined by NMR spectroscopy reveals significant differences compared to those of the typical class I bacterial cytochromes c: a high solvent accessible surface area for the heme group and so-called “intrinsically disordered” nature of the histidine-rich N- and C-terminal regions. Comparison of the signal splitting in the heteronuclear NMR spectra of oxidized, reduced, and TcDH-bound CytC552 reveals the heme axial methionine fluxionality. The TcDH binding site on the CytC552 surface was mapped using NMR chemical shift perturbations. Putative TcDH-CytC552 complexes were reconstructed by the information-driven docking approach and used for the analysis of effective electron transfer pathways. The best pathway includes the electron hopping through His528 and Tyr164 of TcDH, and His83 of CytC552 to the heme group in accordance with pH-dependence of TcDH activity with CytC552

Unusual Cytochrome c552 from Thioalkalivibrio paradoxus: Solution NMR Structure and Interaction with Thiocyanate Dehydrogenase

The search of a putative physiological electron acceptor for thiocyanate dehydrogenase (TcDH) newly discovered in the thiocyanate-oxidizing bacteria Thioalkalivibrio paradoxus revealed an unusually large, single-heme cytochrome c (CytC552), which was co-purified with TcDH from the periplasm. Recombinant CytC552, produced in Escherichia coli as a mature protein without a signal peptide, has spectral properties similar to the endogenous protein and serves as an in vitro electron acceptor in the TcDH-catalyzed reaction. The CytC552 structure determined by NMR spectroscopy reveals significant differences compared to those of the typical class I bacterial cytochromes c: a high solvent accessible surface area for the heme group and so-called “intrinsically disordered” nature of the histidine-rich N- and C-terminal regions. Comparison of the signal splitting in the heteronuclear NMR spectra of oxidized, reduced, and TcDH-bound CytC552 reveals the heme axial methionine fluxionality. The TcDH binding site on the CytC552 surface was mapped using NMR chemical shift perturbations. Putative TcDH-CytC552 complexes were reconstructed by the information-driven docking approach and used for the analysis of effective electron transfer pathways. The best pathway includes the electron hopping through His528 and Tyr164 of TcDH, and His83 of CytC552 to the heme group in accordance with pH-dependence of TcDH activity with CytC552

FGCaMP7, an Improved Version of Fungi-Based Ratiometric Calcium Indicator for In Vivo Visualization of Neuronal Activity

Genetically encoded calcium indicators (GECIs) have become a widespread tool for the visualization of neuronal activity. As compared to popular GCaMP GECIs, the FGCaMP indicator benefits from calmodulin and M13-peptide from the fungi Aspergillus niger and Aspergillus fumigatus, which prevent its interaction with the intracellular environment. However, FGCaMP exhibits a two-phase fluorescence behavior with the variation of calcium ion concentration, has moderate sensitivity in neurons (as compared to the GCaMP6s indicator), and has not been fully characterized in vitro and in vivo. To address these limitations, we developed an enhanced version of FGCaMP, called FGCaMP7. FGCaMP7 preserves the ratiometric phenotype of FGCaMP, with a 3.1-fold larger ratiometric dynamic range in vitro. FGCaMP7 demonstrates 2.7- and 8.7-fold greater photostability compared to mEGFP and mTagBFP2 fluorescent proteins in vitro, respectively. The ratiometric response of FGCaMP7 is 1.6- and 1.4-fold higher, compared to the intensiometric response of GCaMP6s, in non-stimulated and stimulated neuronal cultures, respectively. We reveal the inertness of FGCaMP7 to the intracellular environment of HeLa cells using its truncated version with a deleted M13-like peptide; in contrast to the similarly truncated variant of GCaMP6s. We characterize the crystal structure of the parental FGCaMP indicator. Finally, we test the in vivo performance of FGCaMP7 in mouse brain using a two-photon microscope and an NVista miniscope; and in zebrafish using two-color ratiometric confocal imaging

Novel Genetically Encoded Bright Positive Calcium Indicator NCaMP7 Based on the mNeonGreen Fluorescent Protein

Green fluorescent genetically encoded calcium indicators (GECIs) are the most popular tool for visualization of calcium dynamics in vivo. However, most of them are based on the EGFP protein and have similar molecular brightnesses. The NTnC indicator, which is composed of the mNeonGreen fluorescent protein with the insertion of troponin C, has higher brightness as compared to EGFP-based GECIs, but shows a limited inverted response with an ΔF/F of 1. By insertion of a calmodulin/M13-peptide pair into the mNeonGreen protein, we developed a green GECI called NCaMP7. In vitro, NCaMP7 showed positive response with an ΔF/F of 27 and high affinity (Kd of 125 nM) to calcium ions. NCaMP7 demonstrated a 1.7-fold higher brightness and similar calcium-association/dissociation dynamics compared to the standard GCaMP6s GECI in vitro. According to fluorescence recovery after photobleaching (FRAP) experiments, the NCaMP7 design partially prevented interactions of NCaMP7 with the intracellular environment. The NCaMP7 crystal structure was obtained at 1.75 Å resolution to uncover the molecular basis of its calcium ions sensitivity. The NCaMP7 indicator retained a high and fast response when expressed in cultured HeLa and neuronal cells. Finally, we successfully utilized the NCaMP7 indicator for in vivo visualization of grating-evoked and place-dependent neuronal activity in the visual cortex and the hippocampus of mice using a two-photon microscope and an NVista miniscope, respectively