A Retrospective Population Study of 385 191 Positive Real-Time Reverse Transcription-Polymerase Chain Reaction Tests For SARS-CoV-2 from a Single Laboratory in Katowice, Poland from April 2020 to July 2022.

BACKGROUND This retrospective population study identified 385 191 positive real-time reverse transcription-polymerase chain reaction (RT-PCR) tests for the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) from a single laboratory in Katowice, Poland, from April 2020 to July 2022. MATERIAL AND METHODS The material was nasopharyngeal, nasopharyngeal swab or bronchial lavage, and bronchoalveolar lavage (BAL) to confirm or exclude SARS-CoV-2 infection with the RT-PCR technique. Personal data are use according to the Provisions on the Protection of Personal Data by the Gyn-Centrum laboratory. RESULTS In 9 months of 2020, the number of SARS-CoV-2 results was 88 986; in 2021, it was 168 439, and in the first 7 months of 2022, it was 12 786. In 2020, the highest number of positive results was recorded in the third quarter (83 094 cases); 2021, in the 1st, 2nd, and 4th quarters (58 712; 37 720; and 71 753 cases, respectively), and in 2022, in the 1st quarter (127 613 cases) of the year. A positive result was observed more often in women and people aged 30-39, followed by those 40-49 years. Patients aged 10-19 years comprised the smallest population of SARS-CoV-2-positive cases. CONCLUSIONS In the Polish population studied, from April 2020 to July 2022, the detection rates of SARS-CoV-2 positivity were significantly higher for women than for men and in the 30-49 age group for both sexes. Also, the infection detection rate of 385 191 out of 1 332 659 patient samples, or 28.9%, supports that the Polish society adhered to public health recommendations for infection control during the COVID-19 pandemic.

Reinfections from SARS-CoV-2: A Retrospective Study from the Gyncentrum Genetic Laboratory in Sosnowiec, Poland, April 2020 to July 2022.

BACKGROUND The increasing number of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) reinfections has opened a new research direction related to analyzing long-term immune response and accurately characterizing individual cases of reinfection to understand its mechanism and estimate the risk of widespread reinfection both locally and globally. This retrospective study from the Gyncentrum Genetic Laboratory in Sosnowiec, Poland aimed to evaluate reinfections from SARS-CoV-2 between April 2020 and July 2022. MATERIAL AND METHODS The study extended the previously published report on SARS-CoV-2 infection cases in Poland by analyzing 8041 reinfections diagnosed with real-time reverse transcription-polymerase chain reaction (RT-PCR) assay. Data were collected on the amount of time elapsed from the first infection to the next and, based on these data, all results were divided into several groups for statistical analysis: 0-44, 45-90, 91-200, 201-310, 311-420, and >420 (days). RESULTS The study showed that of the 8041 patients who experienced reinfection, the vast majority (5505) became reinfected more than 310 days after the original infection, even though the average time between infections was 354.3 days. Statistical analysis revealed that the risk of SARS-CoV-2 reinfection increases with time and that this relationship becomes statistically significant after the 200th day following the initial infection (p<0.01). CONCLUSIONS We have shown that acquired immunity to SARS-CoV-2 infection is relatively short-lived - it starts diminishing about 6 months after the initial positive test. Moreover, the risk of reinfection is very high more than 1 year after the initial infection.

Knockdown of the AKT3 (PKBγ), PI3KCA, and VEGFR2 genes by RNA interference suppresses glioblastoma multiforme T98G cells invasiveness in vitro.

Glioblastoma multiforme (GBM) is the most common primary brain malignancy, having a very poor prognosis and is characterized by extensive brain invasion as well as resistance to the therapy. The phosphoinositide 3-kinase (PI3K)/Akt/PTEN signaling pathway is deregulated in GBM. Besides, florid vascularization and aberrantly elevated vascular endothelial growth factor (VEGF) occur very often. The present study was designed to examine the inhibitory effect of AKT3, PI3KCA, and VEGFR2 small interfering RNAs (siRNAs) on GBM cell invasiveness. T98G cells were transfected with AKT3, PI3KCA, and/or VEGFR2 siRNAs. VEGFR2 protein-positive cells were identified by flow cytometry using specific monoclonal anti-VEGFR2 antibodies. Alterations in messenger RNA (mRNA) expression of VEGF, VEGFR2, matrix metalloproteinases (MMPs) (MMP-2, MMP-9, MMP-13, MMP-14), tissue inhibitors of metalloproteinases (TIMPs) (TIMP-1, TIMP-3), c-Fos, c-Jun, hypoxia-inducible factor-1α (HIF-1α), ObRa, and cathepsin D genes were analyzed by qRT-PCR. Cells treated with specific siRNA were also analyzed for invasion using the Matrigel invasion assay. We have found significantly lower mRNA levels of MMPs, cathepsin D, VEGF, VEGFR2, HIF-1α, and c-Fos/c-Jun ratio, as well as significantly higher mRNA level of TIMPs in AKT3 and PI3KCA siRNA transfected cells compared to untransfected cells, while significantly lower mRNA levels of MMPs (MMP-2, MMP-9, MMP-14) and TIMP-1, as well as significantly higher mRNA level of TIMP-3, were shown only in cells transfected with VEGFR2 siRNA. The positive correlation between MMP-13 and ObRa mRNA copy number has been found. Summarizing, transfection of T98G cells with AKT3, PI3KCA, or VEGFR2 siRNAs leads to a significant reduction in cell invasiveness. The siRNA-induced AKT3, PI3KCA, and VEGFR2 mRNA knockdown may offer a novel therapeutic strategy to reduce the invasiveness of GBM cells.

Dermatan Sulfate Affects the Activation of the Necroptotic Effector MLKL in Breast Cancer Cell Lines via the NFκB Pathway and Rac-Mediated Oxidative Stress.

Dermatan sulfate (DS) is a glycosaminoglycan characterized by having a variable structure and wide distribution in animal tissues. We previously demonstrated that some structural variants of DS were able to rapidly induce moderate necroptosis in luminal breast cancer cells when used at a high concentration. We have now investigated the mechanisms underlying the DS-mediated activation of the necroptotic executor MLKL using immunofluorescence, Western blotting and pharmacological inhibition. The two main processes, by which DS influences the phosphorylation of MLKL, are the activation of NFκB, which demonstrates a suppressive impact, and the induction of oxidative stress, which has a stimulatory effect. Moreover, the triggering of the redox imbalance by DS occurs via the modulatory influence of this glycosaminoglycan on the rearrangement of the actin cytoskeleton, requiring alterations in the activity of small Rho GTP-ase Rac1. All of these processes that were elicited by DS in luminal breast cancer cells showed a dependence on the structure of this glycan and the type of cancer cells. Furthermore, our results suggest that a major mechanism that is involved in the stimulation of necroptosis in luminal breast cancer cells by high doses of DS is mediated via the effect of this glycan on the activity of adhesion molecules.

Dermatan Sulfate Affects Breast Cancer Cell Function via the Induction of Necroptosis.

Dermatan sulfate (DS) is widespread in the extracellular matrix (ECM) of animal tissues. This glycosaminoglycan is characterized by a variable structure, which is reflected in the heterogeneity of its sulfation pattern. The sulfate groups are responsible for the binding properties of DS, which determine an interaction profile of this glycan. However, the detailed role of DS in biological processes such as the neoplasm is still poorly understood. The aim of the study was to assess the effects of the structural variants of DS on breast cancer cells. We found that DS isoforms from normal and fibrotic fascia as well as from intestinal mucosa were able to quickly induce oxidative stress in the cytoplasm and affect the mitochondrial function in luminal breast cancer cells. Moreover, the variants caused the necroptosis of the cells most likely via the first of these mechanisms. This death was responsible for a reduction in the viability and number of breast cancer cells. However, the dynamics and intensity of all of the DS variants-triggered effects were strongly dependent on the cell type and the structure of these molecules. The most pronounced activity was demonstrated by those variants that shared structural features with the DS from the tumor niche.

From Alpha to Delta-Genetic Epidemiology of SARS-CoV-2 (hCoV-19) in Southern Poland.

In Poland, the first case of SARS-CoV-2 infection was confirmed in March 2020. Since then, many circulating virus lineages fueled rapid pandemic waves which inflicted a severe burden on the Polish healthcare system. Some of these lineages were associated with increased transmissibility and immune escape. Mutations in the viral spike protein, which is responsible for host cell recognition and serves as the primary target for neutralizing antibodies, are of particular importance. We investigated the molecular epidemiology of the SARS-CoV-2 clades circulating in Southern Poland from February 2021 to August 2021. The 921 whole-genome sequences were used for variant identification, spike mutation, and phylogenetic analyses. The Pango B.1.1.7 was the dominant variant (n = 730, 89.68%) from March 2021 to July 2021. In July 2021, the B.1.1.7 was displaced by the B.1.617.2 lineage with 66.66% in July 2021 and 92.3% in August 2021 frequencies, respectively. Moreover, our results were compared with the sequencing available on the GISAID platform for other regions of Poland, the Czech Republic, and Slovakia. The analysis showed that the dominant variant in the analyzed period was B.1.1.7 in all countries and Southern Poland (Silesia). Interestingly, B.1.1.7 was replaced by B.1.617.2 earlier in Southern Poland than in the rest of the country. Moreover, in the Czech Republic and Slovakia, AY lineages were predominant at that time, contrary to the Silesia region.

The dual role of the glycosaminoglycan chondroitin-6-sulfate in the development, progression and metastasis of cancer.

The remarkable structural heterogeneity of chondroitin sulfate (CS) and dermatan sulfate (DS) generates biological information that can be unique to each of these glycosaminoglycans (GAGs), and changes in their composition are translated into alterations in the binding profiles of these molecules. CS/DS can bind to various cytokines and growth factors, cell surface receptors, adhesion molecules, enzymes and fibrillar glycoproteins of the extracellular matrix, thereby influencing both cell behavior and the biomechanical and biochemical properties of the matrix. In this review, we summarize the current knowledge concerning CS/DS metabolism in the human cancer stroma. The remodeling of the GAG profile in the tumor niche is manifested as a substantial increase in the CS content and a gradual decrease in the proportion between DS and CS. Furthermore, the composition of CS and DS is also affected, which results in a substantial increase in the 6-O-sulfated and/or unsulfated disaccharide content, which is concomitant with a decrease in the 4-O-sulfation level. Here, we discuss the possible impact of alterations in the CS/DS sulfation pattern on the binding capacity and specificity of these GAGs. Moreover, we propose potential consequences of the stromal accumulation of chondroitin-6-sulfate for the progression and metastasis of cancer.

Dermatan sulfate is a player in the transglutaminase 2 interaction network.

Transglutaminase 2 (TG2) is a multifunctional protein that is primarily engaged in cell adhesion/signaling or shows Ca2+-dependent transglutaminase activity in the extracellular space of tissues. This latter action leads to the cross-linking of the extracellular matrix (ECM) proteins. The enhanced extracellular expression of TG2 is associated with processes such as wound healing, fibrosis or vascular remodeling that are also characterized by a high deposition of dermatan sulfate (DS) proteoglycans in the ECM. However, it is unknown whether DS may bind to TG2 or affect its function. Using the plasmon surface resonance method, we showed that DS chains, especially those of biglycan, are good binding partners for TG2. The interaction has some requirements as to the DS structure. The competitive effect of heparin on DS binding to TG2 suggests that both glycosaminoglycans occupy the same binding site(s) on the protein molecule. An occurrence of the DS-TG2 interaction was confirmed by the co-immunoprecipitation of this protein with native decorin that is a DS-bearing proteoglycan rather than with the decorin core protein. Moreover, in vivo DS is responsible for both TG2 binding and the regulation of the location of this protein in the ECM as can be suggested from an increased extraction of TG2 from the human fascia only when an enzymatic degradation of the tissue DS was conducted in the presence of the anti-collagen type I antiserum. In addition, DS with a low affinity for TG2 exerted an inhibitory effect on the protein transamidating activity most probably via the control of the accessibility of a substrate. Our data show that DS can affect several aspects of TG2 biology in both physiological and pathological conditions.

Combination Therapy with AKT3 and PI3KCA siRNA Enhances the Antitumor Effect of Temozolomide and Carmustine in T98G Glioblastoma Multiforme Cells.

BACKGROUND: Glioblastoma multiforme (GBM) is the most malignant and invasive human brain tumor, and it is characterized by a poor prognosis and short survival time. Current treatment strategies for GBM, using surgery, chemotherapy and/or radiotherapy, are ineffective. The PI3K/AKT/PTEN signaling pathway is frequently deregulated in this cancer, and it is connected with regulation of the cell cycle, apoptosis, and autophagy. OBJECTIVES: The current study was undertaken to examine the effect of small interfering RNA (siRNA) targeting the AKT3 and PIK3CA genes on the susceptibility of T98G cells to temozolomide (TMZ) and carmustine (BCNU). METHODS: T98G cells were transfected with AKT3 or PI3KCA siRNA. Transfection efficiency was assessed using flow cytometry and fluorescence microscopy. The influence of AKT3 and PI3KCA siRNA in combination with TMZ and BCNU on T98G cell viability, proliferation, apoptosis, and autophagy was evaluated as well. Alterations in messenger RNA (mRNA) expression of apoptosis-related and autophagy-related genes were analyzed using quantitative reverse transcription polymerase chain reaction (QRT-PCR). RESULTS: Transfection of T98G cells with AKT3 or PI3KCA siRNA and exposure to TMZ and BCNU led to a significant reduction in cell viability, accumulation of subG1-phase cells, and reduction of cells in the S and G2/M phases, as well as induction of apoptosis or necrosis, and regulation of autophagy. CONCLUSION: The siRNA-induced AKT3 and PI3KCA mRNA knockdown in combination with TMZ and BCNU inhibited proliferation and induced apoptosis and autophagy in T98G cells. Thus, knockdown of these genes in combination with TMZ and BCNU may offer a novel therapeutic strategy to more effectively control the growth of human GBM cells, but further studies are necessary to confirm a positive phenomenon for the treatment of GBM.

Methylglyoxal (MGO) inhibits proliferation and induces cell death of human glioblastoma multiforme T98G and U87MG cells.

Glioblastoma multiforme (GBM) is the most malignant and invasive human brain tumor and it is characterized by a poor prognosis and short survival time. Current treatment strategies for GBM using surgery, chemotherapy and/or radiotherapy are ineffective. Thus new therapeutic strategies to target GBM are urgently needed. The effect of methylglyoxal (MGO) on the cell cycle, cell death and proliferation of human GBM cells was investigated. The T98G and U87MG cell lines were cultured in modified EMEM supplemented with 10% fetal bovine serum and maintained at 37°C in a humidified atmosphere of 5% CO2 in air. Cells were exposed to methylglyoxal (0.025mM) per 72h. The influence of MGO on T98G and U87MG cell cycle, proliferation and apoptosis was evaluated as well. Cell cycle phase distribution, proliferation, apoptosis were analyzed by flow cytometry. MGO causes changes in cell cycle and induces accumulation of G1/G0-phase cells and reduced fraction of cells in S and G2/M phases. We have also observed inhibition of cell proliferation and induction of apoptosis in cancer cells. We have also revealed that MGO induces senescence of U87MG but not T98G cells, but further studies are necessary in order to clarify and check mechanism of action of methylglyoxal and it Is a positive phenomenon for the treatment of GBM.

Knockdown of AKT3 and PI3KCA by RNA interference changes the expression of the genes that are related to apoptosis and autophagy in T98G glioblastoma multiforme cells.

BACKGROUND: Glioblastoma multiforme (GBM) is the most malignant and invasive human brain tumor and it is characterized by a poor prognosis and short survival time. The PI3K/AKT/PTEN signaling pathway plays a crucial role in GBM development and it is connected with the regulation of apoptosis and autophagy. Akt is involved in various aspects of cancer cell biology such as cell survival, in addition to both apoptosis and autophagy. The current study was undertaken to examine the effect of the siRNAs that target AKT3 and PI3KCA genes on the apoptosis and autophagy of T98G cells. METHODS: T98G cells were transfected with AKT3 and/or PI3KCA siRNAs. Alterations in the mRNA expression of apoptosis- and autophagy-related genes were analyzed using QRT-PCR. LC3IIA protein-positive cells were identified using flow cytometry with specific antibodies. RESULTS: Our findings demonstrate for the first time that the siRNAs that target AKT3 and PI3KCA change the expression of the genes that are related to apoptosis and autophagy and change the expression of the LC3IIA protein in T98G cells. CONCLUSIONS: Thus, there is a high probability that the knockdown of these genes induces apoptosis and autophagy in T98G cells, but further studies are necessary in order to clarify and check whether autophagy induction is a positive phenomenon for the treatment of GBM.

Knockdown of AKT3 (PKBγ) and PI3KCA suppresses cell viability and proliferation and induces the apoptosis of glioblastoma multiforme T98G cells.

Glioblastoma multiforme (GBM) is the most malignant and invasive human brain tumor that is difficult to treat and has a very poor prognosis. Thus, new therapeutic strategies that target GBM are urgently needed. The PI3K/AKT/PTEN signaling pathway is frequently deregulated in a wide range of cancers. The present study was designed to examine the inhibitory effect of AKT3 or PI3KCA siRNAs on GBM cell growth, viability, and proliferation.T98G cells were transfected with AKT3 and/or PI3KCA siRNAs. AKT3 and PI3KCA protein-positive cells were identified using FC and Western blotting. The influence of specific siRNAs on T98G cell viability, proliferation, cell cycle, and apoptosis was evaluated as well using FC. Alterations in the mRNA expression of AKT3, PI3KCA, and apoptosis-related genes were analyzed using QRT-PCR. Knockdown of AKT3 and/or PI3KCA genes in T98G cells led to a significant reduction in cell viability, the accumulation of subG1-phase cells and, a reduced fraction of cells in the S and G2/M phases. Additionally, statistically significant differences in the BAX/BCL-2 ratio and an increased percentage of apoptotic cells were found. The siRNA-induced AKT3 and PI3KCA mRNA knockdown may offer a novel therapeutic strategy to control the growth of human GBM cells.