The clinical course of coronavirus disease 2019 (COVID‐19) varies from mild symptoms to acute respiratory distress syndrome, hyperinflammation, and coagulation disorder. The hematopoietic system plays a critical role in the observed hyperinflammation, particularly in severely ill patients. Using peripheral blood cells (PBCs) for gene expression analysis is valuable to evaluate disease-associated and drug-response related genes. In this study, we aimed to explore the gene expression profile of PBCs in patients with COVID‐19. Material & methods. Whole blood samples were collected from 19 patients with acute COVID‐19 infection and 20 healthy volunteers. The gene expression of PBCs was determined by RT-qPCR. Results. We investigated the expression of cytokines, chemokines, interferon-stimulated pro-oxidation, and coagulation genes in PBCs of the infected and healthy samples. Up-regulated expression of some genes was found out in the blood of COVID‐19-infected patients compared to the healthy sample. Conclusions: We have identified some genes in whole blood that classifies COVID‐19-infected and healthy patients with good accuracy. These results suggested that the expression of cytokines, coagulation, and interferon-stimulated genes in PBCs can be used for early detection of hyperinflammation, coagulation disorders, and evaluation of efficiency treatment of this disease.
Reverse transcriptase of human immunodeficiency virus type 1 is a vitalenzyme in the HIV‐1 replication cycle and an attractive target of attempts to arrest a primary viral infection. We designed a vector for eukaryotic expression of the 66 kDa subunit of reverse transcriptase under the control of the immediate early cytomegalovirus promoter. Efficient transient expression of the 66 kDa subunit of reverse transcriptase was achieved in a variety of cells. Immunostaining of the transfected cells revealed the cytoplasmatic localization of reverse transcriptase. Reverse transcriptase activity was detected in all transfected cell lines. Injection of this plasmid encoding the 66 kDa subunit of reverse transcriptase into mice resulted in strong reverse transcriptase‐specific immune responses indicating that the 66 kDa subunit of reverse transcriptase is expressed in vivo. Sera from DNA‐immunized mice inhibited reverse transcription in vitro.
The deletion of specific genomic sequences is believed to influence the pathogenesis of certain diseases such as cancer. Identification of these sequences could provide novel therapeutic avenues for the treatment of disease. Here, we describe a simple and robust method called cloning of deleted sequences (CODE), which allows the selective cloning of deleted sequences from complex human genomes. Briefly, genomic DNA from two sources (human normal and tumor samples) was digested with restriction enzymes (e.g., BamHI, BglII, and BclI), then ligated to special linkers, and amplified by PCR. Tester (normal) DNA was amplified using a biotinylated primer and dNTPs. Driver (tumor) DNA was amplified using a non-biotinylated primer, but with dUTP instead of dTTP. After denaturation and hybridization, all the driver DNA was destroyed with uracil-DNA glycosylase (UDG), and all imperfect hybrids were digested with mung bean nuclease. Sequences deleted from the driver DNA but present in the tester DNA were purified with streptavidin magnetic beads, and the cycle was repeated three more times. This procedure resulted in the rapid isolation and efficient cloning of genomic sequences homozygously deleted from the driver DNA sample, but present in the tester DNA fraction.
Heparan sulfate proteoglycans (HSPGs) regulate a wide range of biological activities in both physiological and pathological conditions. Altered expression or deregulated function of HSPGs and their heparan sulfate (HS) chains significantly contribute to carcinogenesis as well and crucially depends on the functioning of the complex system of HS biosynthetic/modifying enzymes termed as “GAGosome”. Here, we aimed at investigating the expression profile of the system in a cell culture model of stroma-epithelial crosstalk and searching for transcription factors potentially related to the regulation of expression of the genes involved. Coculture of BjTERT-fibroblasts with normal PNT2 human prostate epithelial cells resulted in significant downregulation (2-4-fold) of transcriptional activity of HS metabolism-involved genes ( EXT1/2, NDST1/2, GLCE, HS2ST1, HS3ST1/2, HS6ST1/2, SULF1/2, HPSE ) in both cell types, whereas coculture with prostate cancer cells (LNCaP, PC3, DU145) demonstrated no significant interchanges. Human Transcription Factor RT 2 Profiler PCR array and manual RT-PCR verification supposed FOS, MYC, E2F, SRF, NR3C1 as potential candidates for regulation and/or coordination of HS biosynthesis. Taken together, transcriptional activity of HS biosynthetic system in normal fibroblasts and prostate epithelial cells during their coculture might be controlled by their intercellular communication, reflecting of adaptation of these cells to each other. The regulation is attenuated or abrogated if normal fibroblasts interact with prostate cancer cells making the cancer cells independent of the limiting effects of fibroblasts, thus contributing to possibility of unlimited growth and progression. Overall, these data demonstrate an ability of cell-cell interactions to affect transcriptional activity of HS biosynthesis-involved genes.
The host range of retroviruses is rather complex and specific. It is controlled by the products of viral structural genes that interact with the determinants both on the surface and within the cell. The possibility to infect and transform duck embryo fibroblasts is shown for the Prague strain of chicken Rous sarcoma virus (subgroup C), though virus production in these cells is restricted. However, after the 6th passage the "adapted" virus gave the titre practically the same as it was for chicken embryo fibroblasts. Provirus of RSV adapted to the duck embryo fibroblasts and integrated into host DNA was isolated from the library of nucleotide sequences of duck embryo fibroblasts transformed by this virus. The nucleotide sequence of such provirus was determined. The alterations in gp85 coding region of the env gene which proved to be the result of recombination with endogeneous RAV-0 sequences were shown. The formation of viral particles with rather high titre was induced by the proviral transfection on both chicken and duck embryo fibroblasts. The contribution of the revealed alterations in the genome of transformation active virus and possible participation of its td mutant in the adaptation to the new host are discussed.
Malignant melanoma is considered to be one of the most aggressive forms of skin cancer - about 80% of skin cancer deaths are associated with this disease. Because melanoma is an immunogenic tumor, effective treatment is based on strategies to enhance the body's immune response. Recent studies have shown that oligoribonucleotides-D-mannitol (ORNs-D-M) complexes possess exhibit in particular antiviral, anti-inflammatory and immunomodulatory actions due to the activation of immune responses. Given the wide range of biological effects, we aimed to investigate the effect of ORNs-D-M on the development of B16 melanoma in mice by controlling tumor growth and genetic analysis of marker genes. Methods. Adult female mice of a C57BL/6J line were used. Suspension of B16 mouse melanoma cell was subcutaneously introduced into the right posterior paw. For the experimental groups, the ORNs-D-M solution (in PBS) was injected once at concentrations of 1.4; 0.7; 0.35; 0.175 mg on animal. The control group received 100 μl of PBS. Tumor size was monitored during the experiment. Expression of T-cell markers (CD3, CD4, CD8, CD247), macrophage markers (CD68, CD163, NOS2), immunotherapy target genes (PDCD1, CD274, CTLA4, CTLA4 / del) and cytokine (IFNB1) were evaluated by real-time qPCR assay in peripheral blood of mice. Results. Our investigations show that different concentrations of ORNs-D-M have the opposite effect on the growth of melanoma B16 tumors. In the group where animals received 1.4 mg of ORNs-D-M, the formation of the solid tumors was not observed; however, in the group with 0.7 mg dose, the average tumor volume was 97% lower than the non-drug group. In the case of lower concentrations, the average tumor size was 2-3 times higher than in the non-drug group. Also, we observed the changes in the expression of major marker genes. The mRNA expression levels of markers of T-cell counts CD3, CD4, CD8, and CD247, in groups with high concentrations of ORNs-D-M approached those of healthy animals. However, in mice, bearing melanoma recorded a decrease in mRNA levels of these genes. As well ORNs-D-M increases in the immune response to cancer and generally decreases the level of immunosuppression, which reflect in the increased expression of CD274, PDCD1, and IFNB1.
Aim: To find putative diagnostic markers for clear cell renal cell carcinomas (ccRCC). Material and methods: Quantitative polymerase chain reaction (Q-PCR), bisulfite treatment, methylation-specific PCR, analysis on cBioPortal for Cancer Genomics. Results: We have found that expression of GPX 1, GPX3, and GPX4 genes was decreased in ccRCC. We have shown that the number of alanine (GCG) repeats at the amino terminus of the GPX1 protein is variable. It was reported earlier that an allele that possess 5 alanine repeats is associated with the increased cancer risk. According to the obtained data, the allele with the 5 alanine repeats was also present in a group of healthy donors. Moreover, the frequency of alleles with repeats was similar among ccRCC patients and healthy individuals. We found that decreased expression of GPXs genes was not associated with promoter methylation. To provide other explanation, an analysis on the gene copy number was performed. We have found the heterozygous deletions for GPX1 gene, amplification for GPX3 gene, and no change in gene copy number for GPX4. Conclusions: Our data support the hypothesis that GPX1, GPX3, and GPX4 genes may play a role in ccRCC cancerogenesis and therefore they might be considered as putative diagnostic markers for ccRCC.
