Первичный умлаут в немецком языке (на материале древних текстов)

Статья из специализированного выпуска научного журнала "Культура народов Причерноморья", материалы которого объединены общей темой "Язык и Мир" и посвящены общим вопросам Языкознания и приурочены к 80-летию со дня рождения Николая Александровича Рудякова.Стаття із спеціалізованого випуску наукового журналу "Культура народов Причерноморья", матеріали якого поєднані загальною темою "Мова і Світ" і присвячені загальним питанням мовознавства і приурочені до 80-річчя з дня народження Миколи Олександровича Рудякова

The TH1 cell lineage-determining transcription factor T-bet suppresses TH2 gene expression by redistributing GATA3 away from TH2 genes

Lineage-determining transcription factors (LD-TFs) drive the differentiation of progenitor cells into a specific lineage. In CD4+ T cells, T-bet dictates differentiation of the TH1 lineage, whereas GATA3 drives differentiation of the alternative TH2 lineage. However, LD-TFs, including T-bet and GATA3, are frequently co-expressed but how this affects LD-TF function is not known. By expressing T-bet and GATA3 separately or together in mouse T cells, we show that T-bet sequesters GATA3 at its target sites, thereby removing GATA3 from TH2 genes. This redistribution of GATA3 is independent of GATA3 DNA binding activity and is instead mediated by the T-bet DNA binding domain, which interacts with the GATA3 DNA binding domain and changes GATA3′s sequence binding preference. This mechanism allows T-bet to drive the TH1 gene expression program in the presence of GATA3. We propose that redistribution of one LD-TF by another may be a common mechanism that could explain how specific cell fate choices can be made even in the presence of other transcription factors driving alternative differentiation pathways

IMP1 KH1 and KH2 domains create a structural platform with unique RNA recognition and re-modelling properties

IGF2 mRNA-binding protein 1 (IMP1) is a key regulator of messenger RNA (mRNA) metabolism and transport in organismal development and, in cancer, its mis-regulation is an important component of tumour metastasis. IMP1 function relies on the recognition of a diverse set of mRNA targets that is mediated by the combinatorial action of multiple RNA-binding domains. Here, we dissect the structure and RNA-binding properties of two key RNA-binding domains of IMP1, KH1 and KH2, and we build a kinetic model for the recognition of RNA targets. Our data and model explain how the two domains are organized as an intermolecular pseudo-dimer and that the important role they play in mRNA target recognition is underpinned by the high RNA-binding affinity and fast kinetics of this KH1KH2-RNA recognition unit. Importantly, the high-affinity RNA-binding by KH1KH2 is achieved by an inter-domain coupling 50-fold stronger than that existing in a second pseudo-dimer in the protein, KH3KH4. The presence of this strong coupling supports a role of RNA re-modelling in IMP1 recognition of known cancer targets

Capsid-like particles decorated with the SARS-CoV-2 receptor-binding domain elicit strong virus neutralization activity

The rapid development of a SARS-CoV-2 vaccine is a global priority. Here, we develop two capsid-like particle (CLP)-based vaccines displaying the receptor-binding domain (RBD) of the SARS-CoV-2 spike protein. RBD antigens are displayed on AP205 CLPs through a split-protein Tag/Catcher, ensuring unidirectional and high-density display of RBD. Both soluble recombinant RBD and RBD displayed on CLPs bind the ACE2 receptor with nanomolar affinity. Mice are vaccinated with soluble RBD or CLP-displayed RBD, formulated in Squalene-Water-Emulsion. The RBD-CLP vaccines induce higher levels of serum anti-spike antibodies than the soluble RBD vaccines. Remarkably, one injection with our lead RBD-CLP vaccine in mice elicits virus neutralization antibody titers comparable to those found in patients that had recovered from COVID-19. Following booster vaccinations, the virus neutralization titers exceed those measured after natural infection, at serum dilutions above 1:10,000. Thus, the RBD-CLP vaccine is a highly promising candidate for preventing COVID-19.Molecular basis of virus replication, viral pathogenesis and antiviral strategie

Bispecific T cell-engager targeting oncofetal chondroitin sulfate induces complete tumor regression and protective immune memory in mice

Abstract Background The malaria protein VAR2CSA binds oncofetal chondroitin sulfate (ofCS), a unique chondroitin sulfate, expressed on almost all mammalian cancer cells. Previously, we produced a bispecific construct targeting ofCS and human T cells based on VAR2CSA and anti-CD3 (V-aCD3Hu). V-aCD3Hu showed efficacy against xenografted tumors in immunocompromised mice injected with human immune cells at the tumor site. However, the complex effects potentially exerted by the immune system as a result of the treatment cannot occur in mice without an immune system. Here we investigate the efficacy of V-aCD3Mu as a monotherapy and combined with immune checkpoint inhibitors in mice with a fully functional immune system. Methods We produced a bispecific construct consisting of a recombinant version of VAR2CSA coupled to an anti-murine CD3 single-chain variable fragment. Flow cytometry and ELISA were used to check cell binding capabilities and the therapeutic effect was evaluated in vitro in a killing assay. The in vivo efficacy of V-aCD3Mu was then investigated in mice with a functional immune system and established or primary syngeneic tumors in the immunologically “cold” 4T1 mammary carcinoma, B16-F10 malignant melanoma, the pancreatic KPC mouse model, and in the immunologically “hot” CT26 colon carcinoma model. Results V-aCD3Mu had efficacy as a monotherapy, and the combined treatment of V-aCD3Mu and an immune checkpoint inhibitor showed enhanced effects resulting in the complete elimination of solid tumors in the 4T1, B16-F10, and CT26 models. This anti-tumor effect was abscopal and accompanied by a systemic increase in memory and activated cytotoxic and helper T cells. The combined treatment also led to a higher percentage of memory T cells in the tumor without an increase in regulatory T cells. In addition, we observed partial protection against re-challenge in a melanoma model and full protection in a breast cancer model. Conclusions Our findings suggest that V-aCD3Mu combined with an immune checkpoint inhibitor renders immunologically “cold” tumors “hot” and results in tumor elimination. Taken together, these data provide proof of concept for the further clinical development of V-aCD3 as a broad cancer therapy in combination with an immune checkpoint inhibitor

Cryo-EM reveals the architecture of placental malaria VAR2CSA and provides molecular insight into chondroitin sulfate binding

Placental malaria can have severe consequences for both mother and child and effective vaccines are lacking. Parasite-infected red blood cells sequester in the placenta through interaction between parasite-expressed protein VAR2CSA and the glycosaminoglycan chondroitin sulfate A (CS) abundantly present in the intervillous space. Here, we report cryo-EM structures of the VAR2CSA ectodomain at up to 3.1 Å resolution revealing an overall V-shaped architecture and a complex domain organization. Notably, the surface displays a single significantly electropositive patch, compatible with binding of negatively charged CS. Using molecular docking and molecular dynamics simulations as well as comparative hydroxyl radical protein foot-printing of VAR2CSA in complex with placental CS, we identify the CS-binding groove, intersecting with the positively charged patch of the central VAR2CSA structure. We identify distinctive conserved structural features upholding the macro-molecular domain complex and CS binding capacity of VAR2CSA as well as divergent elements possibly allowing immune escape at or near the CS binding site. These observations will support rational design of second-generation placental malaria vaccines

Capture and Detection of Circulating Glioma Cells Using the Recombinant VAR2CSA Malaria Protein

Diffuse gliomas are the most common primary malignant brain tumor. Although extracranial metastases are rarely observed, recent studies have shown the presence of circulating tumor cells (CTCs) in the blood of glioma patients, confirming that a subset of tumor cells are capable of entering the circulation. The isolation and characterization of CTCs could provide a non-invasive method for repeated analysis of the mutational and phenotypic state of the tumor during the course of disease. However, the efficient detection of glioma CTCs has proven to be challenging due to the lack of consistently expressed tumor markers and high inter- and intra-tumor heterogeneity. Thus, for this field to progress, an omnipresent but specific marker of glioma CTCs is required. In this article, we demonstrate how the recombinant malaria VAR2CSA protein (rVAR2) can be used for the capture and detection of glioma cell lines that are spiked into blood through binding to a cancer-specific oncofetal chondroitin sulfate (ofCS). When using rVAR2 pull-down from glioma cells, we identified a panel of proteoglycans, known to be essential for glioma progression. Finally, the clinical feasibility of this work is supported by the rVAR2-based isolation and detection of CTCs from glioma patient blood samples, which highlights ofCS as a potential clinical target for CTC isolation.Medicine, Faculty ofOther UBCNon UBCUrologic Sciences, Department ofReviewedFacult

Additional file 1 of Bispecific T cell-engager targeting oncofetal chondroitin sulfate induces complete tumor regression and protective immune memory in mice

Additional file 1: Sup. Fig. 1. (A) ELISA showing binding of V-aCD3Mu (Coupled)(Kd = 38.8, Bmax = 3.31), rVAR2 (Kd and Bmax not determined), and V-aCD3Mu (Fused)(Kd = 14.2, Bmax = 3.36) to CSPG on a decorin backbone. Data is representative of a minimum of two separate experiments. (B) Solid 4T1 tumors 50-100 mm3 in size were treated with either PBS (n=5), V-aCD3Mu (Coupled) + CpG (n=8), or V-aCD3Mu (Fused) + CpG (n=8) on day 10, 12, 14, and 17 after tumor injection. Numbers in parentheses indicate the number of animals with complete tumor regression out of all mice in the group. Sup. Fig. 2. (A) Gating strategy on splenocytes and PBMCs in flow cytometry used to determine binding of rVAR2, aCD3Mu, V-aCD3Mu, aCD3Hu, and anti-V5 antibodies to T cells and non-T cell splenocytes/PBMCs. The gating is single cells lymphocytes live cells CD4+ and/or CD8+ cells as T cells and CD4-CD8- cells as non-T cells. The geometric MFI of the anti-penta-HIS antibodies conjugated to Alexa Flour 488 was then used to evaluate the binding of the HIS-tagged proteins. (B) Binding of aCD3Mu (Kd = 4.96, Bmax = 1.05), rVAR2 (Kd = NR, Bmax = 0.46), and V-aCD3Mu (Kd = 1.24, Bmax = 3.38) to murine recombinant CD3 in ELISA with aCD4Mu as a negative control (left). Means and standard deviations are shown. Right pane shows CSA inhibition of binding at 120 nM (right). Each dot represents one data point. Sup. Fig. 3. Cytokines measured from 4T1 and splenocyte co-culture supernatants using ELISA. Mouse splenocytes were incubated with 4T1 cancer cells together with 200 nM of the indicated protein. Sup. Fig. 4. (A) Survival curves for mice with indicated tumors treated as described in Fig. 4. The cut-off for all Kaplan-Meier plots is a tumor volume of $$\ge$$ ≥ 400 mm3. Mice were censored if they had to be excluded from the study prematurely due to reasons other than tumor size. Log-rank test was used for statistical analysis. *p < 0.05. (B) Bioluminescence in vivo imaging of C57BL/6 mice following orthotopic implantation of 5x104 Luciferase+ primary pancreatic cancer cells (CHX2000) derived from KPC mice (LSL-KrasG12D/+; p53f/f; Pdx1-Cre). Sup. Fig. 5. (A-C) Survival curves for mice treated as described in Fig. 5. The cut-off for all Kaplan-Meier plots is a tumor volume of $$\ge$$ ≥ 400 mm3. Mice were censored if they had to be excluded from the study prematurely due to reasons other than tumor size. Log-rank test was used for statistical analysis. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. Sup. Fig. 6. (A) Treatment schedule until day 14 when spleens and tumors were harvested for flow cytometry and the subsequent gating strategy on splenocytes to evaluate different cell types in C-D. (B) Percentage of live cells relative to the PBS group in the spleen. Both CD8+ and CD4+ T cells that are CD69+, CD44hi, CD8+CD25+, or Tregs are shown. (C) UMAPs of splenocytes from all four treatment groups with clustering performed in ClusterExplorer. Cell types in clusters are explained below. Statistics were performed using one-way ANOVA with Dunnett’s post hoc test for comparison of all treatment groups to the PBS group. P values are indicated if significant or important for reading the figure. (D) Correlations between the tumor size and �8+CD69+ (p=0.67) and �4+CD69+ (p=0.14) of all live single cells in the tumor evaluated by simple linear regression. Sup. Fig. 7. Binding of mouse antibodies to 4T1 cells and B16-F10 cells in flow cytometry. Serum from C57BL/6 mice treated as described in materials and methods was diluted as illustrated on the figure and incubated with 200.000 4T1 or B16-F10 cells. Soluble CSA was added if indicated for 1 hour before detection with an anti-mouse IgG antibody conjugated to FITC