Non-adherent cells are difficult to transfect with chemical-mediated delivery methods. Electroporation is an attractive strategy to transfer the molecules of interest into suspension cells. Care must be taken with the viability of the transfected cells since parameters, which increase cell membrane permeability, subsequently increase transfection efficiency, leading to higher cell death indices. We intended to evaluate the distribution of hard-to-transfect UT-7 cells among different subpopulations: transfected/viable, untransfected/viable, transfected/dead, and untransfected/dead populations, for a better understanding of the relation between gene electrotransfer efficacy and cell death. The following electroporation parameters were tested: pulse strength, duration, plasmid DNA concentration, and ZnSO₄ as DNase inhibitor. BTX T820 square-wave generator was used, and 48 h after electroporation, cells were observed for viability and fluorescence analysis. Increasing pulse strength correlated directly with an increased ratio of pEGFP-positive cells and inversely with cell viability. The best results, representing 21% pEGFP positive/viable cells, were obtained after EP with 1 HV 1400 V/cm pulse of 250 µs duration using 200 µg/mL plasmid concentration. Results demonstrated that plasmid concentration played the most significant role in pEGFP electrotransfer into UT-7 cells. These results can represent a relevant improvement of gene electrotransfer to obtain genetically modified suspension cells for further downstream experiments.

Myeloproliferative neoplasms (MPNs) are characterized by increased proliferation of myeloid lineages in the bone marrow. Calreticulin (CALR) 52 bp deletion and CALR 5 bp insertion have been identified in essential thrombocythemia (ET) and primary myelofibrosis (PMF). There is not much data on the crosstalk between mutated CALR and MPN-related signaling pathways, such as JAK/STAT, PI3K/Akt/mTOR, and Hedgehog. Calreticulin, a multifunctional protein, takes part in many cellular processes. Nevertheless, there is little data on how mutated CALR affects the oxidative stress response and oxidative stress-induced DNA damage, apoptosis, and cell cycle progression. We aimed to investigate the role of the CALR 52 bp deletion and 5 bp insertion in the pathogenesis of MPN, including signaling pathway activation and functional analysis in CALR-mutated cells. Our data indicate that the JAK/STAT and PI3K/Akt/mTOR pathways are activated in CALR-mutated cells, and this activation does not necessarily depend on the CALR and MPL interaction. Moreover, it was found that CALR mutations impair calreticulin function, leading to reduced responses to oxidative stress and DNA damage. It was revealed that the accumulation of G2/M-CALR-mutated cells indicates that oxidative stress-induced DNA damage is difficult to repair. Taken together, this study contributes to a deeper understanding of the specific molecular mechanisms underlying CALR-mutated MPNs.

Introduction: Patients with Philadelphia-negative (Ph-) myeloproliferative neoplasms: primary myelofibrosis, essential thrombocythemia and polycythemia vera; often develop thrombotic events. It was suggested that the genetic variants that are responsible for blood coagulation and elevated homocysteine level play causal role in the occurrence of thrombosis. Our aim was to evaluate the single nucleotide polymorphisms in PROS1, EPCR, PROC, MTHFR, MS genes and their associations with the risk of developing thrombosis as well as clinical characteristics in patients with myeloproliferative disorders.Material and methods: The screening of PROS1 g.66847T>C, EPCR c.4678G>C, EPCR c.6936A>G, PROC c.565C>T, MTHFR c.677C>T, MTHFR c.1298A>C, MS c.2756A>G polymorphisms was performed at Lithuanian University of Health Sciences, Kaunas, Lithuania. The PCR-RFLP method was applied.Results: After genotyping 88 patients with Ph- myeloproliferative disorders, the association was found between venous thrombosis and MTHFR c.1298A>C (p=0.019). Regression analysis revealed that carriers of PROS1 66847TC, EPCR 4678GC, 6936AG or GG, PROC 565CT or TT, MTHFR 677CT, MS 2756AG genotypes were associated with a lower risk of developing venous thrombosis. EPCR 6936AG genotype could be considered as a protective factor against arterial thrombosis. Genotypes PROC 565CT and 565TT were associated with a lower risk of decreased levels of MPV, 565TT carriers were less likely to develop arterial or venous thrombosis, compared with those carrying the CC or CT genotype.Conclusions: It can be concluded that more research into these polymorphisms needs to be performed, since there are many conflicting results published regarding the complexity of the possible interactions between these gene variants and predisposition to thrombotic events.

Myeloproliferative neoplasms (MPNs) are clonal hematopoietic disorders characterized by excessive proliferation of one or more myeloid lineages, frequently accompanied by an elevated risk of thrombotic events. Matrix metalloproteinases (MMPs), a family of zinc-dependent endopeptidases, are implicated in numerous inflammatory and vascular pathophysiological processes. In this study, we analyzed the association between selected MMP polymorphisms, rs1799750, rs243865, rs3025058, rs3918242, and rs17576, and thrombotic risk as well as clinical characteristics in patients with MPNs. Genotyping was performed using the polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method. Among the polymorphisms analyzed, a statistically significant association was identified between the MMP-9 rs3918242 CT genotype and an increased risk of arterial thrombosis (OR = 4.206, CI 1.337–13.234, p = 0.014). Moreover, rs3918242 CT was associated with thrombotic events (both arterial and venous thrombosis combined), suggesting a potential contributory role in the prothrombotic phenotype observed in MPNs (OR = 3.200, CI 1.110–9.258, p = 0.031). These findings indicate that genetic variation in MMP-9, particularly rs3918242, may serve as a predictive marker for vascular complications in MPN patients. Further studies with larger cohorts are warranted to confirm these associations and to elucidate the molecular mechanisms underlying the contribution of MMP polymorphisms to thrombosis in MPNs.