imagine (Institute of molecular genetics and genetic engineering)
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Identification and Classification of Novel Genetic Variants: En Route to the Diagnosis of Primary Ciliary Dyskinesia
Get PDFPrimary ciliary dyskinesia (PCD) is a disease caused by impaired function of motile cilia. PCD mainly affects the lungs and reproductive organs. Inheritance is autosomal recessive and X-linked. PCD patients have diverse clinical manifestations, thus making the establishment of proper diagnosis challenging. The utility of next-generation sequencing (NGS) technology for diagnostic purposes allows for better understanding of the PCD genetic background. However, identification of specific disease-causing variants is difficult. The main aim of this study was to create a unique guideline that will enable the standardization of the assessment of novel genetic variants within PCD-associated genes. The designed pipeline consists of three main steps: (1) sequencing, detection, and identification of genes/variants; (2) classification of variants according to their effect; and (3) variant characterization using in silico structural and functional analysis. The pipeline was validated through the analysis of the variants detected in a well-known PCD disease-causing gene (DNAI1) and the novel candidate gene (SPAG16). The application of this pipeline resulted in identification of potential disease-causing variants, as well as validation of the variants pathogenicity, through their analysis on transcriptional, translational, and posttranslational levels. The application of this pipeline leads to the confirmation of PCD diagnosis and enables a shift from candidate to PCD disease-causing gene
Biomarkers of Uremic Cardiotoxicity
Get PDFCardiovascular (CV) morbidity and mortality increase along with the progression of chronic kidney disease (CKD). The potential novel biomarkers of cardiotoxicity have been tested with the aim of the early detection of patients at high CV risk, and among them are markers of inflammation, oxidative stress, acute renal injury, and microRNAs. The study analyzed biomarkers in non-dialysis-dependent (NDD; stage 3a-4 CKD) and dialysis-dependent (DD) CKD patients. The prospective cohort study included 87 patients who were followed for 18 months, during which period newly occurred CV events were recorded. Cox regression analysis confirmed serum albumin, urea, interventricular septum thickness diameter (IVST), the use of calcium antagonist, and erythropoiesis-stimulating agent to be significant predictors of CV outcome. No significant difference was observed in biomarkers of inflammation, oxidative stress, acute kidney injury (IL-18, CRP, ferritin, IMA, SOD, NGAL, and KIM-1), and miR-133a, in regards to the presence/absence of CV event, CV death, and left ventricular hypertrophy. Serum albumin, urea, IVST, and the use of calcium antagonist and erythropoiesis-stimulating agents were confirmed to be factors associated with CV events in CKD patients. Apart from traditional risk factors, new research is needed to define novel and reliable biomarkers of cardiotoxicity in CKD patients
SMAD7 and SMAD4 expression in colorectal cancer progression and therapy response
No full textInhibitory SMAD7 and common mediator SMAD4 play crucial roles in SMAD-dependent TGF-I3 signaling that is often disrupted in colorectal cancer (CRC). This study aimed to profile the expression of SMAD7 and SMAD4 in primary and metastatic CRC and to evaluate their significance in disease progression and therapy response. The expression of SMAD7 and SMAD4 genes was analyzed by quantitative real-time PCR in tissues from 35 primary and metastatic CRC patients and in vitro in 7 human cell lines originating from colon tissue. Expression levels of SMAD7 and SMAD4, as well as their ratio, were determined and their association with tumor characteristics and response to therapy were evaluated. SMAD4 level was significantly lower in tumors compared to non-tumor tissues in both primary (p = 0.001) and metastatic (p = 0.001) CRC patients, while tumor expression of SMAD7 was significantly lower from non-tumor tissue only in metastatic patients (p = 0.017). SMAD7/SMAD4 ratio was elevated in CRC primary tumor tissues and cell lines compared to corresponding non-tumor tissues and cell line, respectively (p = 0.003). SMAD7 expression was significantly elevated in primary tumor tissues obtained from responders to neoadjuvant chemoradiotherapy (nCRT) compared to non-responders (p = 0.014). Alterations of expression and ratio of SMAD7 and SMAD4 in CRC cell lines, primary rectal cancer, and liver metastasis emphasize the importance of these genes in different stages of disease progression. Differential expression of SMAD7 in responders versus non-responders to nCRT should be further investigated for its potential predictive value
Inhibition of miR-21 Promotes Cellular Senescence in NT2-Derived Astrocytes
No full textAstrocytes are the main homeostatic cells in the central nervous system (CNS) that provide mechanical, metabolic, and trophic support to neurons. Disruption of their physiological role or acquisition of senescence-associated phenotype can contribute to the CNS dysfunction and pathology. However, molecular mechanisms underlying the complex physiology of astrocytes are explored insufficiently. Recent studies have shown that miRNAs are involved in the regulation of astrocyte function through different mechanisms. Although miR-21 has been reported as an astrocytic miRNA with an important role in astrogliosis, no link between this miRNA and cellular senescence of astrocytes has been identified. To address the role of miR-21 in astrocytes, with special focus on cellular senescence, we used NT2/A (astrocytes derived from NT2/D1 cells). Downregulation of miR-21 expression in both immature and mature NT2/A by the antisense technology induced the arrest of cell growth and premature cellular senescence, as indicated by senescence hallmarks such as increased expression of cell cycle inhibitors p21 and p53 and augmented senescence-associated beta-galactosidase activity. Additionally, in silico analysis predicted many of the genes, previously shown to be upregulated in astrocytes with the irradiation-induced senescence, as miR-21 targets. Taken together, our results point to miR-21 as a potential regulator of astrocyte senescence. To the best of our knowledge, these are the first data showing the link between miR-21 and cellular senescence of astrocytes. Since senescent astrocytes are associated with different CNS pathologies, development of novel therapeutic strategies based on miRNA manipulation could prevent senescence and may improve the physiological outcome
Probiotic-mediated p38 MAPK immune signaling prolongs the survival of Caenorhabditis elegans exposed to pathogenic bacteria
No full textThe host-microbiota cross-talk represents an important factor contributing to innate immune response and host resistance during infection. It has been shown that probiotic lactobacilli exhibit the ability to modulate innate immunity and enhance pathogen elimination. Here we showed that heat-inactivated probiotic strain Lactobacillus curvatus BGMK2-41 stimulates immune response and resistance of the Caenorhabditis elegans against Staphylococcus aureus and Pseudomonas aeruginosa. By employing qRT-PCR and western blot analysis we showed that heat-inactivated BGMK2-41 activated PMK-1/p38 MAPK immunity pathway which prolongs the survival of C. elegans exposed to pathogenic bacteria in nematode killing assays. The C. elegans pmk-1 mutant was used to demonstrate a mechanistic basis for the antimicrobial potential of BGMK2-41, showing that BGMK2-41 upregulated PMK-1/p38 MAPK dependent transcription of C-type lectins, lysozymes and tight junction protein CLC-1. Overall, this study suggests that PMK-1/p38 MAPK-dependent immune regulation by BGMK2-41 is essential for probiotic-mediated C. elegans protection against gram-positive and gram-negative bacteria and could be further explored for development of probiotics with the potential to increase resistance of the host towards pathogens
Copper(II) and Zinc(II) Complexes with the Clinically Used Fluconazole: Comparison of Antifungal Activity and Therapeutic Potential
Get PDFCopper(II) and zinc(II) complexes with clinically used antifungal drug fluconazole (fcz), {[CuCl2(fcz)(2)](.)5H(2)O}(n), 1, and {[ZnCl2(fcz)(2)]Greek ano teleia2C(2)H(5)OH}(n), 2, were prepared and characterized by spectroscopic and crystallographic methods. The polymeric structure of the complexes comprises four fluconazole molecules monodentately coordinated via the triazole nitrogen and two chlorido ligands. With respect to fluconazole, complex 2 showed significantly higher antifungal activity against Candida krusei and Candida parapsilosis. All tested compounds reduced the total amount of ergosterol at subinhibitory concentrations, indicating that the mode of activity of fluconazole was retained within the complexes, which was corroborated via molecular docking with cytochrome P450 sterol 14 alpha-demethylase (CYP51) as a target. Electrostatic, steric and internal energy interactions between the complexes and enzyme showed that 2 has higher binding potency to this target. Both complexes showed strong inhibition of C. albicans filamentation and biofilm formation at subinhibitory concentrations, with 2 being able to reduce the adherence of C. albicans to A549 cells in vitro. Complex 2 was able to reduce pyocyanin production in Pseudomonas aeruginosa between 10% and 25% and to inhibit its biofilm formation by 20% in comparison to the untreated control. These results suggest that complex 2 may be further examined in the mixed Candida-P. aeruginosa infections
Intrinsically disordered protein AtDSS1(V) participates in plant defense response to oxidative stress
No full textDSS1 is a small protein, highly conserved across different species. As a member of the intrinsically disordered protein family, DSS1 interacts with different protein partners, thus forming complexes involved in diverse biological mechanisms: DNA repair, regulation of protein homeostasis, mRNA export, etc. Additionally, DSS1 has a novel intriguing role in the post-translational protein modification named DSSylation. Oxidatively damaged proteins are targeted for removal with DSS1 and then degraded by proteasome. Yet, DSS1 involvement in the maintenance of genome integrity through homologous recombination is the only function well studied in Arabidopsis research. The fact that animal DSS1 shows wide multifunctionality imposes a need to investigate the additional roles of two Arabidopsis thaliana DSS1 homologs. Having in mind the universality of various biological processes, we considered the possibility of plant DSS1 involvement in cellular homeostasis maintenance during stress exposure. Using real-time PCR and immunoblot analysis, we investigated the profiles of DSS1 gene and protein expression under oxidative stress. We grew and selected the homozygous Arabidopsis mutant line, carrying the T-DNA intron insertion in the DSS1(V) gene. The mutant line was phenotypically described during plant development, and its sensitivity to oxidative stress was characterized. This is the first report which indicates that plant DSS1 gene expression has an altered profile under the influence of oxidative stress. dss1(V)(-/-) plants showed an increased sensitivity to oxidative stress, germinated faster than WT, but generally showed developmental delay in further stages. Our results indicate that the DSS1 protein could be a crucial player in the molecular mechanisms underlying plant abiotic stress responses
Use of seed priming to improve Cd accumulation and tolerance in Silene sendtneri, novel Cd hyper-accumulator
Get PDFChanges in the environment as a result of industrialisation and urbanisation impact negatively on plant growth and crop production. Cadmium (Cd) is one of the most dangerous metals that enters the food chain, with toxic effects on plants and human health. This study evaluated the potential of Silene sendtneri as a novel hyperaccumulator and the role of seed priming in tolerance and accumulation rate of Cd. The effect of different priming agents on germination performance, root growth, seedling development, metal uptake and accumulation, antioxidant defences including enzymatic and non-enzymatic antioxidants has been assessed. Seed priming using silicic acid, proline alone or in combination with salicylic acidenhanced germination, seedling development, and root growth under Cd stress. The same priming treatments induced an increase of water content in shoots and roots when plants were exposed to Cd. The enzymatic antioxidant response was specific for the priming agent used. An increase in ferulic acid and rutin in shoots was related to the increase of Cd concentration in the medium. The concentration of malic and oxalic acid increased significantly in shoots of plants grown on high Cd concentrations compared to low Cd concentrations. Silene sendtneri can accumulate significant levels of Cd with enhanced accumulation rate and tolerance when seeds are primed. The best results are obtained by seed priming using 1% silicic acid, proline and salicylic acid
Prognostic Significance of CDH1, FN1 and VIM for Early Recurrence in Patients with Colorectal Liver Metastasis After Liver Resection
Get PDFPurpose: There are limited data on expression of epithelial-mesenchymal transition (EMT) markers in patients with colorectal liver metastases (CRLM). The study aim was to evaluate the expression and prognostic significance of E-cadherin (CDH1), fibronectin (FN1) and vimentin (VIM) in patients with CRLM after curative-intent liver resection. Patients and Methods: Thirty patients with CRLM managed by curative-intent liver resection were included in this prospective pilot study. Blood samples, colorectal liver metastases and surrounding non-tumor liver tissue were collected. Expression of CDH1, FN1 and VIM was analyzed by quantitative real-time polymerase chain reaction. Expression in CRLM and non-tumor liver tissue was compared, while expression in serum was correlated with CRLM expression. One-year recurrence-free survival was compared between patients with low and high CDH1, FN1 and VIM expression. Results: The expression of CDH1 was similar in CRLM and non-tumor liver tissues, while FN1 and VIM expression was significantly lower in metastatic tissue (P=0.003 and pP lt 0.001, respectively). Serum expression of CDH1 and VIM was detected in 66.7% and 93.3% of patients, respectively, while FN1 was not detected in any of the patients. The correlation of CDH1 and VIM expression between CRLM and serum was not statistically significant. Decreased CDH1 expression in CRLM and decreased VIM expression in serum were associated with early recurrence after surgical treatment of CRLM. Conclusion: Lower expression of CDH1 in CRLM and lower serum expression of VIM were found to be associated with early recurrence after liver resection for CRLM
Pharmacogenomics and pharmacotranscriptomics of glucocorticoids in pediatric acute lymphoblastic leukemia
Get PDFPharmacogenomics and pharmacotranscriptomics contribute to more efficient and safer treatment of many diseases, especially malignancies. Acute lymphoblastic leukemia (ALL) is the most common hematological malignancy during childhood. Glucocorticoids, prednisone and dexamethasone, represent the basis of chemotherapy in pediatric ALL. Therapy causes side effects in 75% of patients and 1–3% of pediatric ALL patients die because of therapy side effects rather than the disease itself. Due to this fact, pharmacogenomics and pharmacotranscriptomics have gained key positions in this field. There is a growing knowledge of pharmacogenomics and pharmacotranscriptomics markers relevant for the success of the glucocorticoid treatment of children with ALL. New technologies, such as next-generation sequencing (NGS) have created a possibility for designing panels of pharmacogenomics and pharmacotranscriptomics markers related to the response to glucocorticoid drugs. Optimization of these panels through population pharmacogenomic studies leads to new knowledge that could open the doors widely to pre-emptive pharmacogenomic testing