International Journal of Molecular Sciences, Apr 13, 2023
This article is an open access article distributed under the terms and conditions of the Creative... more This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY
At the end of 2020, population-based vaccination programs with new generation mRNA-based vaccines... more At the end of 2020, population-based vaccination programs with new generation mRNA-based vaccines began almost all over the world. The aim of the study was to evaluate the titer of anti-SARS-CoV-2 IgG antibodies against the S1 subunit of the virus's spike protein as a marker of the humoral response in 477 patients and the concentration of gamma interferon as an indicator of a cellular response in 28 individuals. In our studies, we used serological enzyme-linked immunosorbent assays. IgG was measured in weeks 2 and 3 after the rst dose and 1-5 weeks after the second dose of an mRNA vaccine in seropositive and seronegative individuals as well as in symptomatic and asymptomatic convalescents. High levels of antibodies were observed in 98% of our vaccinated cohort, and the presence of protective T cells was con rmed in the blood samples of all participants. The humoral immune response is diversi ed and is visible as early as 2-3 weeks after the rst dose of the mRNA vaccine. The level of protection increased signi cantly after the second dose, with the increase being much greater in pre-vaccine healthy subjects and less in convalescents. In the second and third weeks after the second dose, the concentration of IgG antibodies was the highest, and in the following weeks, it decreased gradually. Regular serological measurements on eight subjects show that antibody titers are lower four months after vaccination than before the second dose.
International Journal of Molecular Sciences, Oct 23, 2022
This article is an open access article distributed under the terms and conditions of the Creative... more This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY
This article is an open access article distributed under the terms and conditions of the Creative... more This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY
Array-Based Genomic and Transcriptomic Profiling in Pediatric Acute Leukemias in Search of Genes Responsible for Cyclophosphamide Resistance
Blood, Dec 6, 2014
INTRODUCTION: Cyclophosphamide (CTX) is an alkylating agent of the nitrogen mustard type, regarde... more INTRODUCTION: Cyclophosphamide (CTX) is an alkylating agent of the nitrogen mustard type, regarded as one of the most potent immunosuppressive drugs available. An activated form of this drug, phosphoramide mustard, alkylates, or binds, to DNA. Its cytotoxic effect is mainly due to cross-linking of strands of DNA and RNA, and to inhibition of protein synthesis. Its has been shown that CTX suppress T-helper cell functions with prolonged reduction of B cells due to the slower rate of recovery of B lymphocytes from an alkylating agent. As in many chemotherapeutic drugs, a major hindrance to the effectiveness of cyclophosphamide in long term leukemic therapy is the target cells subsequent development of resistance against the drug. OBJECTIVE: The main objective of the research was to implement the determinations of the ex vivo resistance to cyclophosphamide profile and to identify the genetic profile for pediatric patients with acute leukemias. METHODS: In order to determine the ex vivo drug resistance profile, MTT cytotoxicity assay was performed on mononuclear cells. Gene expression profiles were prepared on the basis of cRNA hybridization to oligonucleotide arrays of the human genome (Affymetrix) for 51 patients with ALL and 16 patients with AML. Hierarchical clustering, assignment location and biological function were performed during the correlation analysis for identified probe sets. Verification of the relative expression level of selected genes was carried out by real time PCR. In order to gain new insights into the molecular mechanisms involved in cyclophosphamide resistance, we performed array-based comparative genomic hybridization (aCGH) on a series of 24 primary acute leukemias, using a SurePrint G3 Human CGH Microarray, 8x60K (Agilent). Data was analyzed by bioinformatics tools: Partek Genomics Suite, PANTHER tools, KEGG Pathway, Agilent Feature Extraction & CytoGenomics. RESULTS: Based on the global expression profile and LC50 values we found, that cyclophosphamide and bortezomid are demonstrating the most different resistances profile, in relation to 20 antileukemic drugs analyzed in the entire research. We observed a multitude of differentially expressed genes, e.g., AKR1C3 (Fold Change=2,81, p-value=0,049), ANXA1 (FC=3,04, p=0,011), BCL2A1 (FC=2.69, p=0,019), SERPINA1 (FC=2.12, p=0.014), DHRS7 (FC=2.13, p=0.005), PCDH9 (FC=-4.58, p=0.015), TTC28 (FC=-2.25, p=0.033) and DUSP1; (FC=-2.91, p=0,002). We assigned the differentially expressed genes to functional pathways (Table 1). Table 2 shows consistently amplified and deleted regions in acute leukemias samples. A 0.29-Mb stable deleted region involving 10 genes was detected at 10q23.31. Among the…
Purpose Besides conventional kidney diseases diagnostics, micro RNAs (miRNAs) assessment in urine... more Purpose Besides conventional kidney diseases diagnostics, micro RNAs (miRNAs) assessment in urine and serum is considered to be a promising non-invasive method of diagnostics of renal parenchymal diseases and valuable therapeutic target also. The purpose of the study was to investigate the role of several miRNAs as a markers of kidney damage. Methods Assessment of 45 chronic kidney disease (CKD) patients stage 1–4 and 17 healthy control. Sample of urine and blood was taken from each participant for molecular analysis using Real Time PCR method to identify such micro-RNAs as: hsa-miR-155-5p, hsa-miR-214-3p, hsa-miR-200a-5p, hsa-miR-29a-5p, hsa-miR-21-5p, hsa-miR-93-5p, and hsa-miR-196a-5p. Basic biochemical test was done. Analysis was performed in CKD patients group and subgroup with chronic glomerulonephritis (CGN) confirmed by kidney biopsy. Moreover, analysis was performed in subgroup with different estimated glomerular filtration rate (eGFR) (according to CKD–EPI equation: eGFR &...
Journal of Analytical & Pharmaceutical Research, 2021
Introduction: The main objective was to implement the determinations of the ex vivo resistance to... more Introduction: The main objective was to implement the determinations of the ex vivo resistance to cyclophosphamide and to identify the genetic profile for pediatric patients with acute leukemias. Methods: In order to determine the ex vivo drug resistance profile, MTT cytotoxicity assay was performed on mononuclear cells. Gene expression profiles were prepared on the basis of cRNA hybridization to oligonucleotide arrays of the human genome (Affymetrix). We performed also array-based comparative genomic hybridization using a SurePrint G3 Human CGH Microarray. Data was analyzed by bioinformatics tools. Verification of the relative expression level of 20 genes was carried out by qRT- PCR. Results: We observed a multitude of differentially expressed genes, e.g. ANXA1 (FC=3,04), BCL2A1 (FC=2,69), SERPINA1 (FC=2,12), DHRS7 (FC=2,13), PCDH9 (FC=- 4,58), TTC28 (FC=-2,25) and DUSP1 (FC=-2,91). The expression of genes that code for inflammation mediated by chemokine and cytokine signaling, Wnt...
Biotechnologia. Journal of Biotechnology, Computational Biology and Bionanotechnology, 2013
Eukaryotic genes typically contain introns that are removed post-transcriptionally from the precu... more Eukaryotic genes typically contain introns that are removed post-transcriptionally from the precursor mRNA (pre-mRNA) through splicing. The presence of numerous exons per gene enables the splicing machinery to process the same pre-mRNA differently by selectively joining different exons, generating different transcripts from a single gene via a process named alternative splicing. In contrast to transcriptional control, alternative splicing can influence almost all aspects of protein function and has emerged as a key mechanism for generating proteome diversity and functional complexity. Its prevalence in many genomes, including those of higher plants, suggests that alternative splicing plays crucial roles in biological processes, as is emphasized by the fact that its misregulation can lead to many human diseases. However, information on the functional significance of this posttranscriptional regulation mechanism in plant systems is surprisingly scarce. We have identified an Arabidopsis thaliana gene, ZIFL1 , encoding a membrane transporter from the Major Facilitator Superfamily that plays important roles in both root auxin transport and drought stress tolerance. Selection of an alternative 3N splice site in the ZIFL1 pre-mRNA generates two splice variants that differ in only two nucleotides. While the longer transcript encodes the full-length transporter, the shorter contains a premature stop codon and codes for a truncated protein lacking the 67 C-terminal amino acids. Sequencing, promoter-reporter gene and fluorescent protein fusion experiments indicate that the full-length protein localizes specifically at the tonoplast of root cells, whereas the C-terminal truncation targets the transporter to the plasma membrane of stomatal guard cells. Using reverse genetics, we show that the root tonoplast-localized transporter regulates various auxin-related processes, while the truncated protein mediates drought tolerance by regulating stomatal closure. Heterologous expression in yeast revealed that the two splice forms share proton-coupled potassium transport activity. Thus, by determining the subcellular and tissue localization of two isoforms, alternative splicing allows the same gene to fulfill two very different but equally relevant roles in the plant.
Kluczowymi enzymami zaangażowanymi w metabolizm cząsteczek hormonu i utrzymanie homeostazy w rośl... more Kluczowymi enzymami zaangażowanymi w metabolizm cząsteczek hormonu i utrzymanie homeostazy w roślinie są: 20-oksydazy giberelinowe (ang. gibberellin 20-oxidases, GA20ox), 3-oksydazy giberelinowe (ang. gibberellin 3-oxidases, GA3ox) i 2-oksydazy giberelinowe (ang. gibberellin 2-oxidases, GA-2ox). Podczas gdy GA20ox i GA3ox, katalizując utlenianie odpowiednio 20. i 3. atomu węgla w cząsteczkach GA, nadają im aktywność biologiczną, GA2ox odpowiadają za ich dezaktywację (Olszewski i współaut. 2002). Odkryto również dwa alternatywne sposoby zmniejszania zawartości hormonu w tkankach: epoksydację, za którą odpowiedzialny jest enzym EUI (ang. elongated uppermost internode), zidentyfikowany po raz pierwszy u ryżu (Oryza sativa) (zhu i współaut. 2006) oraz metylację, którą katalizują metylotransferazy giberelinowe GAMT1 i GAMT2 (ang. gibberellins methyltransferases 1/2) zidentyfikowane u rzodkiewnika (Arabidopsis thaliana) (VarbanOwa i współaut. 2007). Zastosowanie w ostatnich kilkunastu latach nowoczesnych technik biologii molekularnej doprowadziło do określenia wzorców ekspresji genów kodujących białka odpowiedzialne za metabolizm GA, co pośrednio umożliwiło oznaczenie miejsc syntezy i degradacji GA oraz poznanie mechanizmów Prawidłowy rozwój roślin zależy zarówno od czynników endogennych, jak i egzogennych, a ich zintegrowanie jest niezwykle ważne w celu zapewnienia właściwego przebiegu wszystkich procesów wzrostowo-rozwojowych. Wewnętrzna kontrola odbywa się głównie z udziałem fitohormonów, wśród których wyróżniamy gibereliny (GA). W ontogenezie roślin GA wywierają istotny wpływ m. in. na kiełkowanie nasion, wzrost wydłużeniowy hipokotyli i międzywęźli, indukcję kwitnienia, rozwój liści i kwiatów oraz dojrzewanie owoców (DaVies 2004, Fleet i sun 2005). Z ponad 130 różnych GA zidentyfikowanych u roślin, grzybów i bakterii (GA 1-GA 136 , http://www.plant-hormones.info/ga1info.htm), tylko nieliczne (GA 1 , GA 3 , GA 4 , GA 5 , GA 6 , GA 7) wykazują aktywność biologiczną, natomiast pozostałe są ich prekursorami lub produktami katabolizmu (MacMillan 2002). Pierwsze etapy biosyntezy GA są wspólne dla wszystkich terpenoidów. Kolejne reakcje utleniania (m. in. grupy aldehydowej do karboksylowej, oksydacyjnej dekarboksylacji, hydroksylacji pierścienia, odwodorowania z utworzeniem podwójnego wiązania) oraz dehydratacji, z utworzeniem dodatkowego pierścienia laktonowego, prowadzą do powstania wszystkich przedstawicieli rodziny GA (Ryc. 1) (kOpcewicz i lewak 2002).
Biotechnologia. Journal of Biotechnology, Computational Biology and Bionanotechnology, 2013
Eukaryotic genes typically contain introns that are removed post-transcriptionally from the precu... more Eukaryotic genes typically contain introns that are removed post-transcriptionally from the precursor mRNA (pre-mRNA) through splicing. The presence of numerous exons per gene enables the splicing machinery to process the same pre-mRNA differently by selectively joining different exons, generating different transcripts from a single gene via a process named alternative splicing. In contrast to transcriptional control, alternative splicing can influence almost all aspects of protein function and has emerged as a key mechanism for generating proteome diversity and functional complexity. Its prevalence in many genomes, including those of higher plants, suggests that alternative splicing plays crucial roles in biological processes, as is emphasized by the fact that its misregulation can lead to many human diseases. However, information on the functional significance of this posttranscriptional regulation mechanism in plant systems is surprisingly scarce. We have identified an Arabidopsis thaliana gene, ZIFL1 , encoding a membrane transporter from the Major Facilitator Superfamily that plays important roles in both root auxin transport and drought stress tolerance. Selection of an alternative 3N splice site in the ZIFL1 pre-mRNA generates two splice variants that differ in only two nucleotides. While the longer transcript encodes the full-length transporter, the shorter contains a premature stop codon and codes for a truncated protein lacking the 67 C-terminal amino acids. Sequencing, promoter-reporter gene and fluorescent protein fusion experiments indicate that the full-length protein localizes specifically at the tonoplast of root cells, whereas the C-terminal truncation targets the transporter to the plasma membrane of stomatal guard cells. Using reverse genetics, we show that the root tonoplast-localized transporter regulates various auxin-related processes, while the truncated protein mediates drought tolerance by regulating stomatal closure. Heterologous expression in yeast revealed that the two splice forms share proton-coupled potassium transport activity. Thus, by determining the subcellular and tissue localization of two isoforms, alternative splicing allows the same gene to fulfill two very different but equally relevant roles in the plant.
Here we determined the impact of salt shock and salt stress on the level of DNA methylation in se... more Here we determined the impact of salt shock and salt stress on the level of DNA methylation in selected CpG islands localized in promoters or first exons of sixteen salt-responsive genes in beets. Two subspecies differing in salt tolerance were subjected for analysis, a moderately salt-tolerant sugar beetBeta vulgaris ssp.vulgariscv. Huzar and a halophytic beet,Beta vulgaris ssp.maritima. The CpG island methylation status was determined. All target sequences were hyper- or hypomethylated under salt shock and/or salt stress in one or both beet subspecies. It was revealed that the genomic regions analyzed were highly methylated in both, the salt treated plants and untreated controls. Methylation of the target sequences changed in a salt-dependent manner, being affected by either one or both treatments. Under both shock and stress, the hypomethylation was a predominant response in sugar beet. InBeta vulgaris ssp.maritima, the hypermethylation occurred with higher frequency than hypomet...
The increase of human population and associated increasing demand for agricultural products lead ... more The increase of human population and associated increasing demand for agricultural products lead to soil over-exploitation. Biofertilizers based on lyophilized plant material containing living plant growth-promoting microorganisms (PGPM) could be an alternative to conventional fertilizers that fits into sustainable agricultural technologies ideas. We aimed to: (1) assess the diversity of endophytic bacteria in sugar and sea beet roots and (2) determine the influence of osmoprotectants (trehalose and ectoine) addition during lyophilization on bacterial density, viability and salt tolerance. Microbiome diversity was assessed based on 16S rRNA amplicons sequencing, bacterial density and salt tolerance was evaluated in cultures, while bacterial viability was calculated by using fluorescence microscopy and flow cytometry. Here we show that plant genotype shapes its endophytic microbiome diversity and determines rhizosphere soil properties. Sea beet endophytic microbiome, consisting of ge...
Osteoarthritis (OA) is a degenerative disease of the joints, characterized by irreversible destru... more Osteoarthritis (OA) is a degenerative disease of the joints, characterized by irreversible destruction of articular cartilage. The disease process is accompanied by changes of immunological nature, resulting in local inflammatory reactions, with the production of proinflammatory cytokines and metalloproteinases. There is currently no effective treatment resulting in repair of degraded cartilage. Clinical application of mesenchymal cells (MSCs) creates new possibilities in the treatment of incurable diseases. Multipotent MSCs exhibit immunosuppressive activity and limited immunogenicity and have the potential to differentiate in vitro towards adipocytes, osteocytes, chondrocytes, myocytes and endothelial cells. Thanks to these biological properties, they are increasingly used in clinical therapies. In few scientific papers, the safety of cellular therapies in the group of dogs diagnosed with OA has been confirmed. In patients undergoing treatment with autologous intra-articular injec...
Background: Sugar beet is a highly salt-tolerant crop. However, its ability to withstand high sal... more Background: Sugar beet is a highly salt-tolerant crop. However, its ability to withstand high salinity is reduced compared to sea beet, a wild ancestor of all beet crops. The aim of this study was to investigate transcriptional patterns associated with physiological, cytological and biochemical mechanisms involved in salt response in these closely related subspecies. Salt acclimation strategies were assessed in plants subjected to either gradually increasing salt levels (salt-stress) or in excised leaves, exposed instantly to salinity (salt-shock). Result: The majority of DEGs was down-regulated under stress, which may lead to certain aspects of metabolism being reduced in this treatment, as exemplified by lowered transpiration and photosynthesis. This effect was more pronounced in sugar beet. Additionally, sugar beet, but not sea beet, growth was restricted. Silencing of genes encoding numerous transcription factors and signaling proteins was observed, concomitantly with the up-regulation of lipid transfer protein-encoding genes and those coding for NRTs. Bark storage protein genes were up-regulated in sugar beet to the level observed in unstressed sea beet. Osmotic adjustment, manifested by increased water and proline content, occurred in salt-shocked leaves of both genotypes, due to the concerted activation of genes encoding aquaporins, ion channels and osmoprotectants synthesizing enzymes. bHLH137 was the only TF-encoding gene induced by salt in a dose-dependent manner irrespective of the mode of salt treatment. Moreover, the incidence of bHLH-binding motives in promoter regions of salinity-regulated genes was significantly greater than in non-regulated ones. Conclusions: Maintaining homeostasis under salt stress requires deeper transcriptomic changes in the sugar beet than in the sea beet. In both genotypes salt shock elicits greater transcriptomic changes than stress and it results in greater number of up-regulated genes compared to the latter. NRTs and bark storage protein may play a yet undefined role in salt stress-acclimation in beet. bHLH is a putative regulator of salt response in beet leaves and a promising candidate for further studies.
Identification of the Genomic Rearrangements Associated with the Ex Vivo Resistance to Anthracyclines in Childhood Acute Leukemias
Blood, 2015
INTRODUCTION: Resistance to drugs is connected with several genomic changes, but the major mutati... more INTRODUCTION: Resistance to drugs is connected with several genomic changes, but the major mutations are still unknown. Daunorubicin (DNR), idarubicin (IDA), doxorubicin (DOX) and mitoxantrone (MIT) are chemotherapeutics of the anthracycline family that are commonly used to treat acute leukemias (AL). OBJECTIVE: The aim of the study was to identify changes on the genome level and compare them with ex vivo resistance to DNR, IDA, DOX and MIT among children diagnosed with acute lymphoblastic (ALL) or myeloblastic (AML) leukemia. METHODS: The in vitro drug resistance profile was determined in MTT cytotoxicity assay, which was performed on mononuclear cells taken from 44 (aCGH) and 187 (qPCR) patients. The cyanine labeled genomic DNA (Cy3 - reference, Cy5 - patient) was hybridized to comparative genomic hybridization array (SurePrint G3 Human CGH Microarray 8x60K, Agilent). Data were analyzed using bioinformatics tools, like: Feature Extraction and CytoGenomics (Agilent) and databases, ...
Whole Genome and Transcriptome Analysis of Genetic Alterations in Context of Busulfan in Vitro Resistance in Pediatric Acute Leukemias
Blood, 2015
INTRODUCTION: Busulfan (BUS) is bifunctional cell cycle non-specific alkylating antineoplastic ag... more INTRODUCTION: Busulfan (BUS) is bifunctional cell cycle non-specific alkylating antineoplastic agent. High-dose busulfan in combination with cyclophosphamide is an effective preparative regimen for patients undergoing allogeneic or autologous bone marrow transplantation for leukemias and solid tumors. The main mechanism of action involves the interference of DNA replication and RNA transcription (by alkylation and cross-linking of strands), which results in the disruption of nucleic acid functions. Alterations in drug transport and metabolism, increased activity of glutathione S-transferase and aldehyde dehydrogenase activity or enhanced DNA repair may also play a role in the resistance to this alkylator. OBJECTIVE: The aim of this study was to elucidate candidate genes and molecular pathways involved in ex vivo resistance to busufan in childhood acute leukemias. METHODS: In order to determine the in vitro BUS resistance profile, MTT cytotoxicity assay was performed on mononuclear c...
This study found that extracts containing ionically bound proteins, isolated from potato pulp and... more This study found that extracts containing ionically bound proteins, isolated from potato pulp and brewers' spent grain, were characterized by high peroxidase activities. The kinetic parameters, namely K m and V max values, were typical for plant peroxidases. Seven peroxidase isoenzymes in potato pulp and two isoenzymes in brewers' spent grain were obtained from their respective ionically bound fractions. Peroxidases from both potato pulp and brewers' spent grain displayed high storage stability, over a 90-day-long storage period, if stored at 2208C with glycerol added to a concentration of 50% or as unsupplemented extracts at 48C. Peroxidase activity was present in the covalently bound fraction of potato pulp, whereas it was absent in the respective fraction of brewers' spent grain. Covalently bound peroxidases from potato pulp displayed high activity, but low stability. Peroxidases extracted from brewers' spent grain and potato pulp, followed the pingpong mechanism and the sequence mechanism, respectively. Practical applications Plant peroxidases are widely applicable in various fields of biotechnology and diagnostics. These enzymes are used for biosensor and glucometer construction and in conjugation with antibodies for the purpose of enzyme immunoassays. Using waste products as a peroxidase source provides a cost-friendly alternative to commercially available horseradish peroxidase and gives an opportunity to recycle waste from the food industry-processed plant biomass. Here, the properties of peroxidases from food waste products, namely potato pulp and brewers' spent grain are analyzed. The extracts were characterized by high storage stability and high enzymatic activity, which are two key traits necessary for the practical use of enzyme preparations.
Translational Research in Veterinary Science, 2018
MicroRNAs are a class of small, evolutionarily conserved, endogenous RNAs, capable of controlling... more MicroRNAs are a class of small, evolutionarily conserved, endogenous RNAs, capable of controlling gene expression. MicroRNAs are transcribed by RNA polymerases II and III, generating precursors that undergo a series of cleavage events to form mature microRNA. They play an important regulatory role in animals at the posttranscriptional levels by targeting mRNAs for direct cleavage of mRNAs or repression of mRNA translation. The main biological function of miRNA is the post-translation regulation of cells, like: proliferation and differentiation, cell death, fat metabolism, neuronal patterning and angiogenesis. These molecules are the main regulators of biological features of economic
This is the first communication of micropropagation system for Inula germanica using seedling exp... more This is the first communication of micropropagation system for Inula germanica using seedling explants germinated in vitro. The development of this system gives the possibility of future reintroduction of I. germanica providing a way to stabilize or re-establish its population. Shoot tips and fragments of cotyledons, hypocotyls and roots were isolated from ten-day-old seedlings. Explants were put on MS medium containing 1.0 mg l-1 benzylaminopurine and 0.1 mg l-1 naphthaleneacetic acid and cultured under continuous white fluorescent light (45 μmol.m-2.s-1) at 26 ± 1 °C. The highest percentage of shoot organogenesis (83.3%) was recorded for hypocotyl, while the highest average number of shoots per explant (12.0) was recorded for shoot tips. In subsequent subcultures, multiplication rate decreased to 3.0-4.9 shoots per explant. Less than 19% shoots were able to root on the solid medium without auxins. The highest rooting efficiency (69.3%) was recorded for solid medium supplemented wi...
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