Publications by authors named "Paweł Wołkow"

57 Publications

Status of CHEK2 and p53 in patients with early-onset and conventional gastric cancer.

Oncol Lett 2021 May 3;21(5):348. Epub 2021 Mar 3.

Department of Human Anatomy, Medical University of Lublin, 20-090 Lublin, Poland.

Gastric cancer (GC) is the fourth most common cause of cancer-associated death. Based on the age at diagnosis, GC is divided into early-onset GC (EOGC; ≤45 years) and conventional GC (CGC; >45 years). Mutations in the cell cycle checkpoint kinase 2 () and genes are associated with several types of cancer; however, their genetic defects in GC remain poorly understood. The aim of the present study was to determine the subcellular distribution of the CHEK2 protein and its redistribution following DNA damage, to improve the understanding of the DNA damage response. Genetic alterations and patterns of expression of CHEK2 and p53 proteins were investigated to identify potential biological markers and indicators of GC development. Additionally, the affected signaling pathways and their clinical importance in GC development and associated syndromes were investigated. A total of 196 GC samples (89 CGC and 107 EOGC samples) were used in the present study. DNA from 53 samples (18 CGC and 35 EOGC samples) was sequenced using targeted next-generation sequencing technology to identify and compare common and rare mutations associated with GC. Subsequently, the cytoplasmic and nuclear expression levels of CHEK2, phosphorylated (p)-CHEK2 at threonine 68 and p53 in GC tissues were determined via immunohistochemistry. Sequencing resulted in the identification of 63 single nucleotide polymorphisms (SNPs) in the gene amongst 5 different variants, and the intron variant c.319+379A>G was the most common SNP. In the gene, 57 different alterations were detected amongst 9 variant types, and the missense variant c.215C>G was the most common. Nuclear CHEK2 expression was high in both the EOGC and CGC subtypes. However, the prevalence of cytoplasmic CHEK2 expression (P<0.001) and nuclear p-CHEK2 expression (P=0.011) was significantly higher in CGC compared with in EOGC tissues. There was a statistically significant difference between high and low cytoplasmic CHEK2 expression in patients with p53-positive EOGC compared with in patients with p53-positive CGC (P=0.002). The present study was designed to determine the association between CHEK2 and p53 expression patterns in patients with EOGC and CGC, as well as genetic alterations in the and genes.
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http://dx.doi.org/10.3892/ol.2021.12609DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7967923PMC
May 2021

Low Expression of miR-375 and miR-190b Differentiates Grade 3 Patients with Endometrial Cancer.

Biomolecules 2021 Feb 13;11(2). Epub 2021 Feb 13.

Center for Medical Genomics OMICRON, Jagiellonian University Medical College, 31-034 Krakow, Poland.

Endometrial cancer (EC) is treated according to the stage and prognostic risk factors. Most EC patients are in the early stages and they are treated surgically. However some of them, including those with high grade (grade 3) are in the intermediate and high intermediate prognostic risk groups and may require adjuvant therapy. The goal of the study was to find differences between grades based on an miRNA gene expression profile. Tumor samples from 24 patients with grade 1 ( = 10), 2 ( = 7), and 3 ( = 7) EC were subjected to miRNA profiling using next generation sequencing. The results obtained were validated using the miRNA profile of 407 EC tumors from the external Cancer Genome Atlas (TCGA) cohort. We obtained sets of differentially expressed (DE) miRNAs with the largest amount between G2 to G1 (50 transcripts) and G3 to G1 (40 transcripts) patients. Validation of our results with external data (TCGA) gave us a reasonable gene overlap of which we selected two miRNAs (miR-375 and miR190b) that distinguish the high grade best from the low grade EC. Unsupervised clustering showed a high degree of heterogeneity within grade 2 samples. MiR-375 as well as 190b might be useful to create grading verification test for high grade EC. One of the possible mechanisms that is responsible for the high grade is modulation by virus of host morphology or physiology.
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http://dx.doi.org/10.3390/biom11020274DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7918779PMC
February 2021

The transcriptome-wide association search for genes and genetic variants which associate with BMI and gestational weight gain in women with type 1 diabetes.

Mol Med 2021 01 20;27(1). Epub 2021 Jan 20.

Center for Medical Genomics OMICRON, Jagiellonian University Medical College, Kraków, Poland.

Background: Clinical data suggest that BMI and gestational weight gain (GWG) are strongly interconnected phenotypes; however, the genetic basis of the latter is rather unclear. Here we aim to find genes and genetic variants which influence BMI and/or GWG.

Methods: We have genotyped 316 type 1 diabetics using Illumina Infinium Omni Express Exome-8 v1.4 arrays. The GIANT, ARIC and T2D-GENES summary statistics were used for TWAS (performed with PrediXcan) in adipose tissue. Next, the analysis of association of imputed expression with BMI in the general and diabetic cohorts (Analysis 1 and 2) or GWG (Analysis 3 and 4) was performed, followed by variant association analysis (1 Mb around identified loci) with the mentioned phenotypes.

Results: In Analysis 1 we have found 175 BMI associated genes and 19 variants (p < 10) which influenced GWG, with the strongest association for rs11465293 in CCL24 (p = 3.18E-06). Analysis 2, with diabetes included in the model, led to discovery of 1812 BMI associated loci and 207 variants (p < 10) influencing GWG, with the strongest association for rs9690213 in PODXL (p = 9.86E-07). In Analysis 3, among 648 GWG associated loci, 2091 variants were associated with BMI (FDR < 0.05). In Analysis 4, 7 variants in GWG associated loci influenced BMI in the ARIC cohort.

Conclusions: Here, we have shown that loci influencing BMI might have an impact on GWG and GWG associated loci might influence BMI, both in the general and T1DM cohorts. The results suggest that both phenotypes are related to insulin signaling, glucose homeostasis, mitochondrial metabolism, ubiquitinoylation and inflammatory responses.
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http://dx.doi.org/10.1186/s10020-020-00266-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7818927PMC
January 2021

The Gut Microbiota Profile According to Glycemic Control in Type 1 Diabetes Patients Treated with Personal Insulin Pumps.

Microorganisms 2021 Jan 12;9(1). Epub 2021 Jan 12.

Department of Metabolic Diseases, Jagiellonian University Medical College, 2 Jakubowskiego Street, 30-688 Krakow, Poland.

Recently, several studies explored associations between type 1 diabetes (T1DM) and microbiota. The aim of our study was to assess the colonic microbiota structure according to the metabolic control in T1DM patients treated with insulin pumps. We studied 89 T1DM patients (50.6% women) at the median age of 25 (IQR, 22-29) years. Pielou's evenness ( = 0.02), and Shannon's ( = 0.04) and Simpson's diversity indexes ( = 0.01), were higher in patients with glycosylated hemoglobin (HbA1c) ≥ 53 mmol/mol (7%). There were no differences in beta diversity between groups. A linear discriminant analysis effect size (LEfSe) algorithm showed that one family () was enriched in patients with HbA1c < 53 mmol/mol, whereas one family () and four species (, unclassified species of , , and ) were enriched in patients with HbA1c ≥ 53 mmol/mol. We found that at class level, the following pathways according to Kyoto Encyclopedia of Genes and Genomes were enriched in patients with HbA1c < 53 mmol/mol: bacterial motility proteins, secretion system, bacterial secretion system, ribosome biogenesis, translation proteins, and lipid biosynthesis, whereas in patients with HbA1c ≥ 53 mmol/mol, the galactose metabolism, oxidative phosphorylation, phosphotransferase system, fructose, and mannose metabolism were enriched. Observed differences in alpha diversity, metabolic pathways, and associations between bacteria and HbA1c in colonic flora need further investigation.
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http://dx.doi.org/10.3390/microorganisms9010155DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7826603PMC
January 2021

Dysregulation of Transcription Factor Activity During Formation of Cancer-Associated Fibroblasts.

Int J Mol Sci 2020 Nov 19;21(22). Epub 2020 Nov 19.

Center for Medical Genomics OMICRON, Jagiellonian University Medical College, 31-034 Kraków, Poland.

The reciprocal interactions between cancer cells and the quiescent fibroblasts leading to the activation of cancer-associated fibroblasts (CAFs) serve an important role in cancer progression. Here, we investigated the activation of transcription factors (TFs) in prostate fibroblasts (WPMY cell line) co-cultured with normal prostate or tumorous cells (RWPE1 and RWPE2 cell lines, respectively). After indirect co-cultures, we performed mRNA-seq and predicted TF activity using mRNA expression profiles with the Systems EPigenomics Inference of Regulatory Activity (SEPIRA) package and the GTEx and mRNA-seq data of 483 cultured fibroblasts. The initial differential expression analysis between time points and experimental conditions showed that co-culture with normal epithelial cells mainly promotes an inflammatory response in fibroblasts, whereas with the cancerous epithelial, it stimulates transformation by changing the expression of the genes associated with microfilaments. TF activity analysis revealed only one positively regulated TF in the RWPE1 co-culture alone, while we observed dysregulation of 45 TFs (7 decreased activity and 38 increased activity) uniquely in co-culture with RWPE2. Pathway analysis showed that these 45 dysregulated TFs in fibroblasts co-cultured with RWPE2 cells may be associated with the RUNX1 and PTEN pathways. Moreover, we showed that observed dysregulation could be associated with expression. We conclude that phenotypic changes in fibroblast responses to co-culturing with cancer epithelium result from orchestrated dysregulation of signaling pathways that favor their transformation and motility rather than proinflammatory status. This dysregulation can be observed both at the TF and transcriptome levels.
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http://dx.doi.org/10.3390/ijms21228749DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7699520PMC
November 2020

Next-Generation Sequencing as a Tool to Detect Vaginal Microbiota Disturbances during Pregnancy.

Microorganisms 2020 Nov 18;8(11). Epub 2020 Nov 18.

Department of Molecular Medical Microbiology, Chair of Microbiology, Faculty of Medicine, Jagiellonian University Medical College, 31-121 Krakow, Poland.

The physiological microbiota of the vagina is responsible for providing a protective barrier, but Some factors can disturb the balance in its composition. At that time, the amounts of the genus decrease, which may lead to the development of infection and severe complications during pregnancy. The aim of the study was the analysis of the bacterial composition of the vagina in 32 Caucasian women at each trimester of pregnancy using the next-generation sequencing method and primers targeting V3-V4 regions. In the studied group, the dominant species were and . Statistically significant differences in the quantitative composition between trimesters were observed in relation to , , spp. Out of the 32 patients, 20 demonstrated fluctuations within the genus and 9 of them, at different stages of pregnancy, exhibited the presence of potentially pathogenic microbiota, among others: spp., , and The composition of the vaginal microbiota during pregnancy was subject to partial changes over trimesters. Although in one-third of the studied patients, both the qualitative and quantitative composition of microbiota was relatively constant, in the remaining patients, physiological and potentially pathogenic fluctuations were distinguished.
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http://dx.doi.org/10.3390/microorganisms8111813DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7698737PMC
November 2020

Decreased Expression of the High Mobility Group Box 1 () Gene in Peripheral Blood in Patients with Mild or Moderate Infection.

Microorganisms 2020 Aug 11;8(8). Epub 2020 Aug 11.

Department of Infectious and Tropical Diseases, Jagiellonian University Medical College, 30-688 Krakow, Poland.

Cytokines are mediators of inflammation induced in the course of infection (CDI). High Mobility Group Box 1 (HMGB1) is a cytokine playing an important role in the pathogenesis of numerous inflammatory and autoimmune diseases. The aim of the study was to assess the gene expression in the course of CDI. We have performed a prospective case-control study- including 55 adult patients, among them 27 with CDI, who were hospitalized from October 2018 to February 2020 and 28 healthy volunteers. We assessed: a complete blood count with differential leukocyte count, blood creatinine, albumin, and C-reactive protein (CRP) levels. Then, the expression of the gene was evaluated using quantitative Real-Time PCR. Patients with CDI were found to have a significant increase in white blood cells (WBC), neutrophil count, and CRP levels, they also exhibited decreased levels of albumin compared with controls. The gene expression was significantly lower among patients with CDI compared with the control group and significantly, inversely correlated with CRP level in blood. In conclusion, we have observed a decreased expression of the gene in peripheral blood of patients with mild or moderate CDI, which hypothetically could reflect their diminished capability to fight the pathogen.
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http://dx.doi.org/10.3390/microorganisms8081217DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7464922PMC
August 2020

High-Throughput Sequencing of Gastric Cancer Patients: Unravelling Genetic Predispositions Towards an Early-Onset Subtype.

Cancers (Basel) 2020 Jul 21;12(7). Epub 2020 Jul 21.

Department of Human Anatomy, Medical University of Lublin, 20-090 Lublin, Poland.

Background: Gastric cancer is the fourth most common cause of cancer-related death. Currently, it is broadly accepted that the molecular complexity and heterogeneity of gastric cancer, both inter- and intra-tumor, display important barriers for finding specific biomarkers for the early detection and diagnosis of this malignancy. Early-onset gastric cancer is not as prevalent as conventional gastric carcinoma, but it is a preferable model for studying the genetic background, as young patients are less exposed to environmental factors, which influence cancer development.

Aim: The main objective of this study was to reveal age-dependent genotypic characteristics of gastric cancer subtypes, as well as conduct mutation profiling for the most frequent alterations in gastric cancer development, using targeted next-generation sequencing technology.

Patients And Methods: The study group included 53 patients, consisting of 18 patients with conventional gastric cancer and 35 with an early-onset subtype. The DNA of all index cases was used for next-generation sequencing, employing a panel of 94 genes and 284 single nucleotide polymorphisms (SNPs) (TruSight Cancer Panel, Illumina), which is characteristic for common and rare types of cancer.

Results: From among the 53 samples processed for sequencing, we were able to identify seven candidate genes (, and ) and nine variants among them: one splice_acceptor, four synonymous, and four missense variants. These were selected for the age-dependent differentiation of gastric cancer subtypes. We found four variants with C-Score ≥ 10, as 10% of the most deleterious substitutions: rs1800862 (), rs10138997 (), rs2230009 (), and rs2959656 (). We identified 36 different variants, among 24 different genes, which were the most frequent genetic alterations among study subjects. We found 16 different variants among the genes that were present in 100% of the total cohort: (rs2746462), (rs1670283), (rs2958057), (rs4925828; rs11342077, rs398010167; rs2721190), (rs326212), (rs540012), (rs4930199), (rs659243), (rs1169305), (rs206075; rs169547), (rs9514066; rs9514067), and (rs7183618).

Conclusions: The technology of next-generation sequencing is a useful tool for studying the development and progression of gastric carcinoma in a high-throughput way. Our study revealed that early-onset gastric cancer has a different mutation frequency profile in certain genes compared to conventional subtype.
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http://dx.doi.org/10.3390/cancers12071981DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7409326PMC
July 2020

Mitochondrial GWAS and association of nuclear - mitochondrial epistasis with BMI in T1DM patients.

BMC Med Genomics 2020 07 7;13(1):97. Epub 2020 Jul 7.

Center for Medical Genomics OMICRON, Jagiellonian University Medical College, Kraków, Poland.

Background: BMI is a strong indicator of complications from type I diabetes, especially under intensive treatment.

Methods: We have genotyped 435 type 1 diabetics using Illumina Infinium Omni Express Exome-8 v1.4 arrays and performed mitoGWAS on BMI. We identified additive interactions between mitochondrial and nuclear variants in genes associated with mitochondrial functioning MitoCarta2.0 and confirmed and refined the results on external cohorts: the Framingham Heart Study (FHS) and GTEx data. Linear mixed model analysis was performed using the GENESIS package in R/Bioconductor.

Results: We find a borderline significant association between the mitochondrial variant rs28357980, localized to MT-ND2, and BMI (β = - 0.69, p = 0.056). This BMI association was confirmed on 1889 patients from FHS cohort (β = - 0.312, p = 0.047). Next, we searched for additive interactions between mitochondrial and nuclear variants. MT-ND2 variants interacted with variants in the genes SIRT3, ATP5B, CYCS, TFB2M and POLRMT. TFB2M is a mitochondrial transcription factor and together with TFAM creates a transcription promoter complex for the mitochondrial polymerase POLRMT. We have found an interaction between rs3021088 in MT-ND2 and rs6701836 in TFB2M leading to BMI decrease (inter_pval = 0.0241), while interaction of rs3021088 in MT-ND2 and rs41542013 in POLRMT led to BMI increase (inter_pval = 0.0004). The influence of these interactions on BMI was confirmed in external cohorts.

Conclusions: Here, we have shown that variants in the mitochondrial genome as well as additive interactions between mitochondrial and nuclear SNPs influence BMI in T1DM and general cohorts.
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http://dx.doi.org/10.1186/s12920-020-00752-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7341625PMC
July 2020

Negative pressure wound therapy affects circulating plasma microRNAs in patients with diabetic foot ulceration.

Diabetes Res Clin Pract 2020 Jul 10;165:108251. Epub 2020 Jun 10.

Department of Metabolic Diseases, Jagiellonian University Medical College, Krakow, Poland; University Hospital, Krakow, Poland. Electronic address:

Aims: Negative pressure wound therapy (NPWT) is commonly used in diabetic foot ulceration (DFU). The molecular mechanisms of NPWT action, particularly outside of the wound site, have not been described. We assessed NPWT's effect on circulating miRNA expression levels in type 2 diabetes (T2DM) patients with DFU.

Methods: We examined 34 T2DM patients treated with either NPWT (n = 24) or standard therapy (ST, n = 10). The group assignment was based on clinical criteria and local practice. Next-generation sequencing-based microRNA expression was determined on the patient's plasma collected before therapy and after 8 days.

Results: NPWT patients were similar to the ST group in terms of age, BMI, and HbA1c level; however, they differed by mean wound area (12.6 cm vs. 1.1 cm p = 0.0005). First, we analyzed the change of miRNA after NPWT or ST and observed an upregulation of let-7f-2 only in the NPWT group. Then, we analyzed the differential expression between NPWT and ST groups, looking at possible wound size effects. We found 12 differentially expressed miRNAs in pre-treatment comparison, including let-7f-2, while in post-treatment analysis we identified 28 miRNAs. The pathway enrichment analysis suggests that identified miRNAs may be involved in wound healing, particularly through angiogenesis.

Conclusion: We found initial evidence that NPWT in T2DM patients with DFU affects miRNA expression in plasma. Additionally, some differences in plasma miRNA expression may be related to wound size.
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http://dx.doi.org/10.1016/j.diabres.2020.108251DOI Listing
July 2020

Relations between circulating and myocardial fibrosis-linked microRNAs with left ventricular reverse remodeling in dilated cardiomyopathy.

Adv Clin Exp Med 2020 Mar;29(3):285-293

Department of Cardiac and Vascular Diseases, John Paul II Hospital, Kraków, Poland.

Background: Left ventricular reverse remodeling (LVRR) determines clinical status and outcomes in dilated cardiomyopathy (DCM). The extent of myocardial fibrosis is connected to the systolic function of the heart. The recent discovery of the contribution of microRNAs (miRs) to the regulation of cardiac remodeling, LVRR and fibrosis warrants exploration.

Objectives: The aim of the study was to examine the predictive value of circulating and myocardial miR expression for LVRR in DCM.

Material And Methods: Seventy consecutive DCM patients (age 48 ±12.1 years, 90% male, ejection fraction (EF) 24.4% ±7.4%) were included in the study. At baseline, all patients underwent clinical assessment, echocardiography, venous blood sampling, and right ventricular endomyocardial biopsy. Circulating and myocardial miRs (miR-21, -26, -29, -30, -133a, and -423) were measured with quantitative real-time polymerase chain reaction (qRT-PCR). LVRR was defined as an increase in EF ≥ 10%, accompanied by a decrease in left ventricle end-diastolic diameter (LVEDd) ≥10% or LVEDd ≤ 33 mm/m2 between baseline and 3-month follow-up.

Results: At the 3-month follow-up, 4 patients had died and 3 patients had incomplete data. The remaining patients were divided according to the presence of LVRR into LVRR-present (n = 32, 51%) and LVRR-absent (n = 31, 49%) groups. Out of all the circulating and tissue miRs under study, only myocardial expression of miR-133a significantly differed between the LVRR-present and LVRR-absent group (1.22 (0.47-1.90) vs 0.61 (0.25-0.99) ΔCq, respectively, p < 0.01). miR-133a was found to be a significant LVRR predictor in unadjusted (odds ratio (OR) = 2.81 (1.23-6.40), p < 0.05) and adjusted for duration of disease, left ventricle end-diastolic (LVED) volume (LVEDvol), hs-troponin-T, and NT-proBNP (OR = 5.20 (1.13-24.050, p < 0.05) models.

Conclusions: From all of the circulating and tissue miRs, only myocardial miR-133a showed increased expression in LVRR-present patients and was found an independent LVRR predictor. This indicates a link between miR-133 and cardiac remodeling in DCM.
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http://dx.doi.org/10.17219/acem/115088DOI Listing
March 2020

Changes in the Intestinal Microbiota Are Seen Following Treatment with Infliximab in Children with Crohn's Disease.

J Clin Med 2020 Mar 4;9(3). Epub 2020 Mar 4.

Jagiellonian University Medical College, Faculty of Medicine, Department of Pediatrics, Gastroenterology and Nutrition, 30-633 Kraków, Poland.

The aim of the study was to determine the impact of biological treatment with tumor necrosis factor α antibodies (anti-TNF-α) on the intestinal microbiome of children with severe Crohn's disease (CD) and to evaluate the differences in the intestinal microbiome between patients treated with biological therapy and healthy children. Microbiota composition was analyzed by 16S next-generation sequencing (NGS) and microbial profiles were compared between studied groups. Fifty-four samples (from 18 patients before and after anti-TNF-α induction therapy and 18 healthy children) were used in the sequencing analysis. Shannon's diversity index ( = 0.003, adj. = 0.010) and observed operational taxonomic units (OTUs) ( = 0.007, adj. = 0.015) were different between controls and patients with prior therapy for CD. Statistically significant dissimilarities between beta diversity metrics, indicating distinct community composition across groups, were observed in patients with CD before and after therapy. We did not observe any differences between controls and patients with CD after therapy. Core microbiome analysis at species level showed that 32 species were present only in patients with CD but not in controls. The results show that biological treatment is associated with changes in the intestinal microbiome of patients with CD: these changes result in an intestinal microbiome pattern similar to that seen in healthy children. Long-term observation is necessary to determine whether treatment can lead to full restoration of a healthy-like microbiome.
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http://dx.doi.org/10.3390/jcm9030687DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7141282PMC
March 2020

Epigenetic mechanism in search for the pathomechanism of diabetic neuropathy development in diabetes mellitus type 1 (T1DM).

Endocrine 2020 04 4;68(1):235-240. Epub 2020 Jan 4.

University Hospital in Krakow, Kraków, Poland.

Objective: The aim of this study was to check the hypothesis concerning the crucial role of DNA methylation (one of the epigenetic mechanisms) within selected genes related to the destruction and regeneration of neural cells and its input in the pathogenesis of diabetic neuropathy, using a model of the DNA in peripheral blood cells.

Methods: A cross-sectional, case-control study was conducted, consisting of 24 adult Type 1 Diabetes Melitus (T1DM) patients with autonomic neuropathy (CAN), 25 T1DM patients without neuropathy and 25 matched, healthy adults acting as a control (Ctrl). The Ewing's tests, using the ProSciCard apparatus (Mewicon CATEEM-Tec GmbH), was employed to assess the severity of the patients' symptoms of autonomic neuropathy. For DNA methylation analysis, DNA material of each sample DNA after bisulfite conversion was used for the hybridization of BeadChips (Infinium Methylation EPIC Kit, Illumina), and imaged on the Illumina HiScan. The changes in the expression of selected genes were examined using real-time PCR. Probes were labeled using fluorescein amidite, FAM (Thermo Fisher Scientific). Amplification was performed using the continuous fluorescence detection 7900 HT Fast Real-Time PCR system (Thermo Fisher Scientific). The expression ratio of the target mRNA was normalized to the level of 18s RNA and compared with the control. Statistical analysis was performed using Statistica version 13.1. The statistically significant results were recognized, with a value of p < 0.05.

Results: Clinical analysis of the investigated groups revealed a significantly higher percentage of personal insulin pump users in the group without neuropathy. The glucose metabolic control, based on the HbA1c level analysis, was also significantly better in T1DM patients without CAN. The Bumphunter method for DNA methylation analysis showed statistically significant regions related to the genes involved in nerve regeneration ninjurin 2 (NINJ2) and functionality (BR serine/threonine kinase 2 BRSK2, claudin 4 CLDN4). When compared with T1DM patients without neuropathy, T1DM patients with neuropathy showed significantly increased methylation in the first NINJ2 axon, and a lower level of DNA methylation in the region of the first intron of BRSK2, as well as the CLDN4 5'UTR regions. The qRT-PCR results confirmed the decreased expression of NINJ2 and CLDN4 genes in patients with T1DM with CAN.

Conclusions: The different DNA methylation profiles, correlating with the expression of genes related to nervous tissue development and regeneration in patients with T1DM with autonomic neuropathy provide evidence for the role of epigenetic mechanisms promoting the development of CAN, a chronic complication of T1DM.
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http://dx.doi.org/10.1007/s12020-019-02172-9DOI Listing
April 2020

Differences in the intestinal microbiome of healthy children and patients with newly diagnosed Crohn's disease.

Sci Rep 2019 12 11;9(1):18880. Epub 2019 Dec 11.

Department of Pediatrics, Gastroenterology and Nutrition, Faculty of Medicine, Jagiellonian University Medical College, Wielicka 265, Kraków, 30-663, Poland.

The aetiology of inflammatory bowel diseases (IBD) seems to be strongly connected to changes in the enteral microbiome. The dysbiosis pattern seen in Crohn's disease (CD) differs among published studies depending on patients' age, disease phenotype and microbiome research methods. The aims was to investigate microbiome in treatment-naive paediatric patients to get an insight into its structure at the early stage of the disease in comparison to healthy. Stool samples were obtained from controls and newly diagnosed patients prior to any intervention. Microbiota was analysed by 16SrRNAnext-generation-sequencing (NGS). Differences in the within-sample phylotype richness and evenness (alpha diversity) were detected between controls and patients. Statistically significant dissimilarities between samples were present for all used metrics. We also found a significant increase in the abundance of OTUs of the Enterococcus genus and reduction in, among others, Bifidobacterium (B. adolescentis), Roseburia (R.faecis), Faecalibacterium (F. prausnitzii), Gemmiger (G. formicilis), Ruminococcus (R. bromii) and Veillonellaceae (Dialister). Moreover, differences in alpha and beta diversities in respect to calprotectin and PCDAI were observed: patients with calprotectin <100 µg/g and with PCDAI below 10 points vs those with calprotectin >100 µg/g and mild (10-27.7 points), moderate (27.5-40 points) or severe (>40 points) CD disease activity had higher richness and diversity of gut microbiota. The results of our study highlight reduced diversity and dysbiosis at the earliest stage of the disease. Microbial imbalance and low abundance of butyrate-producing bacteria, including Bifidobacterium adolescentis, may suggest benefits of microbial modification therapy.
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http://dx.doi.org/10.1038/s41598-019-55290-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6906406PMC
December 2019

Contribution of a Novel Variant to Peters Plus Syndrome Discovered by a Combination of Next-Generation Sequencing and Automated Text Mining.

Int J Mol Sci 2019 Nov 28;20(23). Epub 2019 Nov 28.

Center for Medical Genomics-OMICRON, Jagiellonian University Medical College, ul. Kopernika 7c, 31-034 Krakow, Poland.

Anterior segment dysgenesis (ASD) encompasses a spectrum of ocular disorders affecting the structures of the anterior eye chamber. Mutations in several genes, involved in eye development, are implicated in this disorder. ASD is often accompanied by diverse multisystemic symptoms and another genetic cause, such as variants in genes encoding collagen type IV. Thus, a wide spectrum of phenotypes and underlying genetic diversity make fast and proper diagnosis challenging. Here, we used AMELIE, an automatic text mining tool that enriches data with the most up-to-date information from literature, and wANNOVAR, which is based on well-documented databases and incorporates variant filtering strategy to identify genetic variants responsible for severely-manifested ASD in a newborn child. This strategy, applied to trio sequencing data in compliance with ACMG 2015 guidelines, helped us find two compound heterozygous variants of the gene, of which c.660+1G>A (rs80338851) was previously associated with the phenotype of Peters plus syndrome (PPS), while the second, NM_194318.3:c.755delC (p.T252fs), in exon 9 of the same gene was noted for the first time. PPS, a very rare subtype of ASD, is a glycosylation disorder, where the dysfunctional gene product, O-fucose-specific β-1,3-glucosyltransferase, is ineffective in providing a noncanonical quality control system for proper protein folding in cells. Our study expands the mutation spectrum of the gene related to PPS. We suggest that the implementation of automatic text mining tools in combination with careful variant filtering could help translate sequencing results into diagnosis, thus, considerably accelerating the diagnostic process and, thereby, improving patient management.
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http://dx.doi.org/10.3390/ijms20236006DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6928627PMC
November 2019

Cranial Irradiation in Childhood Acute Lymphoblastic Leukemia Is Related to Subclinical Left Ventricular Dysfunction and Reduced Large Artery Compliance in Cancer Survivors.

J Clin Med 2019 Nov 13;8(11). Epub 2019 Nov 13.

Department of Internal Medicine and Gerontology, Jagiellonian University Medical College, 10 Śniadeckich Street, 31-531 Cracow, Poland.

Long-term survivors of acute lymphoblastic leukemia (ALL), the most common childhood malignancy, are at remarkably increased risk of heart failure (HF) in middle age, most likely due anthracycline cardiotoxicity. The role of cranial radiation therapy (CRT) in the development of left ventricular (LV) dysfunction, a predecessor of overt HF, remains unclear. Our aim was to compare LV function and systemic arterial properties according to past CRT in young adult survivors of anthracycline-treated ALL. We studied young adult survivors of childhood ALL at a median of 16 years from diagnosis treated with anthracycline-based chemotherapy, with ( = 12) or without ( = 30) CRT. In addition to fractional shortening (FS) and ejection fraction (EF), LV function was quantified by tissue Doppler imaging of the mitral annulus. Aortic strain/distensibility and arterial compliance were derived from echocardiography and simultaneously recorded pulse pressure. Despite similar FS and EF, peak mitral annular systolic velocity (median (interquartile range): 9.0 (7.5-10.0) vs. 10.0 (8.8-11.5) cm/s, = 0.05), and early diastolic velocity (13.8 (13.0-14.8) vs. 15.5 (14.0-17.3), = 0.01) were decreased after chemotherapy combined with CRT compared to chemotherapy without CRT. Systemic arterial compliance was lower in post-CRT subjects (1.0 (0.8-1.2 vs. 1.4 (1.1-1.7) mL/mmHg, = 0.002). Aortic strain and distensibility were similar regardless of prior CRT. In conclusion, lower arterial compliance and subclinical LV dysfunction may be possible late consequences of past CRT in adult survivors of childhood ALL. Whether arterial stiffening is associated with future HF development in CRT-exposed ALL survivors remains to be investigated.
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http://dx.doi.org/10.3390/jcm8111952DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6912438PMC
November 2019

Clostridium difficile caused changes in fatty acids profile and resolvin D1 content in plasma of infected patients.

Eur J Gastroenterol Hepatol 2020 03;32(3):318-324

Department of Infectious and Tropical Diseases.

Objectives: Clostridium difficile infection (CDI) is an acute gastrointestinal infection caused by anaerobic, toxin-producing bacteria. During the course of CDI, there is a general inflammatory state. In order to gain a deeper understanding of the role of fatty acids (FAs) in the pathogenesis of acute infection we analyzed their plasma content in both patients with CDI and controls.

Methods: The study groups included 40 patients with CDI and 40 healthy volunteers. Plasma FA content was analyzed by gas chromatography, resolvin D1 (RvD1) level using ELISA assay, and we assessed the white blood cell (WBC) count, neutrophil count and C-reactive protein (CRP) level.

Results: Patients with CDI were characterized by significantly higher values of WBC, neutrophils, platelets and CRP compared with the control group. The saturated FA index was statistically higher and total n-3 FA was significantly decreased in the plasma of CDI patients as compared with the control group. RvD1 content was significantly higher in the control group as compared with patients with CDI.

Conclusion: In patients with good outcomes, we probably observed the effective resolution of inflammation, as reflected in n-3 FA metabolism and their significant decrease in plasma. This may indicate the therapeutic role of n-3 FA in CDI infection.
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http://dx.doi.org/10.1097/MEG.0000000000001600DOI Listing
March 2020

Patterns of gene expression characterize T1 and T3 clear cell renal cell carcinoma subtypes.

PLoS One 2019 31;14(5):e0216793. Epub 2019 May 31.

Center for Medical Genomics OMICRON, Medical Faculty, Jagiellonian University Medical College, Krakow, Poland.

Renal carcinoma is the 20th most common cancer worldwide. Clear cell renal cell carcinoma is the most frequent type of renal cancer. Even in patients diagnosed at an early stage, characteristics of disease progression remain heterogeneous. Up-to-date molecular classifications stratify the ccRCC samples into two clusters. We analyzed gene expression in 23 T1 or T3 ccRCC samples. Unsupervised clustering divided this group into three clusters, two of them contained pure T1 or T3 samples while one contained a mixed group. We defined a group of 36 genes that discriminate the mixed cluster. This gene set could be associated with tumor classification into a higher stage and it contained significant number of genes coding for molecular transporters, channel and transmembrane proteins. External data from TCGA used to test our findings confirmed that the expression levels of those 36 genes varied significantly between T1 and T3 tumors. In conclusion, we found a clustering pattern of gene expression, informative for heterogeneity among T1 and T3 tumors of clear cell renal cell carcinoma.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0216793PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6544217PMC
January 2020

Characteristics of gut microbiota in adult patients with type 1 and type 2 diabetes based on next‑generation sequencing of the 16S rRNA gene fragment.

Pol Arch Intern Med 2018 06 15;128(6):336-343. Epub 2018 Apr 15.

Introduction Scientific data indicate a possible influence of gut microbiota on the development of type 1 and type 2 diabetes mellitus (T1DM and T2DM, respectively). Sequence analysis of 16S ribosomal RNA identified several hundred bacterial species of the intestinal ecosystem, most of which cannot be cultured. Objectives We aimed to evaluate gut microbiota composition in adult patients with T1DM and T2DM and establish a link between microbiological test results and patients' clinical data. Patients and methods We examined DNA isolated from fecal samples in 3 groups: healthy volunteers (n = 23), patients with T1DM (n = 22), and patients with T2DM (n = 23). Next‑generation sequencing was performed on the MiSeq platform. Results At the phylum level, the Firmicutes bacteria prevailed (>77%) in all groups. At the taxonomic levels L2 (phylum) and L6 (genus), significant differences were demonstrated in bacterial profiles, particularly in the T2DM group. A negative correlation was observed between several genera of bacteria and the percentage of glycated hemoglobin A1c in the T2DM group, while a positive correlation was revealed between bacteria belonging to the genus Bifidobacterium and high‑density lipoprotein cholesterol levels in both T1DM and T2DM groups. Conclusions Our results provide grounds for conducting research in the field of gut microbiota in order to develop individualized therapy for patients with diabetes based on modifying the microbiota composition, as a new method for controlling glycemia. Next‑generation sequencing allows a rapid identification of the DNA of all bacteria present in the sample and their taxonomic classification.
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http://dx.doi.org/10.20452/pamw.4246DOI Listing
June 2018

In vitro maturation of monocyte-derived dendritic cells results in two populations of cells with different surface marker expression, independently of applied concentration of interleukin-4.

Int Immunopharmacol 2018 Apr 28;57:165-171. Epub 2018 Feb 28.

Jagiellonian University Medical College, Faculty of Medicine, Department of Pharmacology, Ul. Grzegórzecka 16, 31-531 Kraków, Poland.

Dendritic cells (DCs) play a crucial role in the development of adaptive immune response. Monocyte-derived dendritic cells (MDDCs) are generated in vitro to study DC biology and for use in immunotherapy. However, procedures to generate MDDCs vary and an impact this may have on their final phenotype is insufficiently studied. Monocytes isolated from healthy blood donors were cultured for 7 days with granulocyte-macrophage colony stimulating factor (50 ng/mL) and low (500 IU/mL, L-IL4) or high (1000 IU/mL, H-IL4) interleukin 4 (IL4), to obtain immature DCs and for the following 2 days with addition of soluble CD40 ligand (500 ng/mL) and prostaglandin E (1 μg/mL) to obtain mature DCs. We measured mean fluorescence activity and percentage of cells, positive for CD14, HLA-DR, CD80, CD83, CD86, CCR7, and CD1a or CD209 markers after 7 and 9 days of culture, in both IL4 concentrations. Percentage of positively staining mature MDDCs was higher than among immature cells, for all studied markers. Interestingly, varying IL4 concentrations had negligible impact on staining of mature MDDCs. However, immature L-IL4 cultured MDDCs were less intensely stained for HLA-DR and CD209 than H-IL4 immature DCs. Flow cytometry revealed presence of 2 populations of cells (dominant P1 and less prevalent P2), when either L-IL4 or H-IL4 was used. Among mature MDDCs, population P1 had higher percentage of positively staining cells than P2, for all studied markers except CCR7. In conclusion, both concentrations of IL4 produce in vitro heterogeneous populations of mature MDDCs with similar staining for cell surface markers.
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http://dx.doi.org/10.1016/j.intimp.2018.02.015DOI Listing
April 2018

Comparative iTRAQ analysis of protein abundance in the human sinoatrial node and working cardiomyocytes.

J Anat 2018 06 27;232(6):956-964. Epub 2018 Feb 27.

HEART - Heart Embryology and Anatomy Research Team, Department of Anatomy, Faculty of Medicine, Jagiellonian University Medical College, Cracow, Poland.

Our objective was to assess the changes in protein abundance in the human sinoatrial node (SAN) compared with working cardiomyocytes to identify SAN-specific protein signatures. Four pairs of samples (the SAN and working cardiomyocytes) were obtained postmortem from four human donors with no evidence of cardiovascular disease. We performed protein identification and quantitation using two-dimensional chromatography-tandem mass spectrometry with isobaric peptide labeling (iTRAQ). We identified 451 different proteins expressed in both the SAN and working cardiomyocytes, 166 of which were differentially regulated (110 were upregulated in the SAN and 56 in the working cardiomyocytes). We identified sarcomere structural proteins in both tissues, although they were differently distributed among the tested samples. For example, myosin light chain 4, myosin regulatory light chain 2-atrial isoform, and tropomyosin alpha-3 chain levels were twofold higher in the SAN than in working cardiomyocytes, and myosin light chain 3 and myosin regulatory light chain 2-ventricular/cardiac muscle isoform levels were twofold higher in the ventricle tissue than in SAN. We identified many mitochondrial oxidative phosphorylation, β-oxidation, and tricarboxylic acid cycle proteins that were predominantly associated with working cardiomyocytes tissue. We detected upregulation of the fatty acid omega activation pathway proteins in the SAN samples. Some proteins specific for smooth muscle tissue were highly upregulated in the SAN (e.g. transgelin), which indicates that the SAN tissue might act as the bridge between the working myocardium and the smooth muscle. Our results show possible implementation of proteomic strategies to identify in-depth functional differences between various heart sub-structures.
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http://dx.doi.org/10.1111/joa.12798DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5978942PMC
June 2018

The relationship between myocardial fibrosis and myocardial microRNAs in dilated cardiomyopathy: A link between mir-133a and cardiovascular events.

J Cell Mol Med 2018 04 29;22(4):2514-2517. Epub 2018 Jan 29.

Department of Cardiac and Vascular Diseases, John Paul II Hospital, Krakow, Poland.

It is unknown whether fibrosis-associated microRNAs: miR-21, miR-26, miR-29, miR-30 and miR-133a are linked to cardiovascular (CV) outcome. The study evaluated the levels of extracellular matrix (ECM) fibrosis and the prevalence of particular microRNAs in patients with dilated cardiomyopathy (DCM) to investigate any correlation with CV events.

Methods: Seventy DCM patients (48 ± 12 years, EF 24.4 ± 7.4%) underwent right ventricular biopsy. The control group was comprised of 7 patients with CAD who underwent CABG and intraoperative biopsy. MicroRNAs were measured in blood and myocardial tissue via qPCR. The end-point was a combination of CV death and urgent HF hospitalization at the end of 12 months. There were differential levels of circulating and myocardial miR-26 and miR-29 as well as myocardial miR-133a when the DCM and CABG groups were compared. Corresponding circulating and myocardial microRNAs did not correlate with one another. There was no correlation between microRNA and ECM fibrosis. By the end of the 12-month period of the study, CV death had occurred in 6 patients, and a further 19 patients required urgent HF hospitalization. None of the circulating microRNAs was a predictor of the combined end-point; however, myocardial miR-133a was an independent predictor in unadjusted models (HR 1.53; 95% CI 1.14-2.05; P < .004) and adjusted models (HR 1.57; 95% CI 1.14-2.17; P < .005). The best cut-off value for the miR-133a level for the prediction of the combined end-point was 0.74 ΔCq, with an AUC of 0.67. The absence of a correlation between the corresponding circulating and myocardial microRNAs calls into question their cellular source. This study sheds new light on the role of microRNAs in ECM fibrosis in DCM, which warrants further exploration.
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http://dx.doi.org/10.1111/jcmm.13535DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5867126PMC
April 2018

The presence of IL-8 +781 T/C polymorphism is associated with the parameters of severe Clostridium difficile infection.

Microb Pathog 2018 Jan 2;114:281-285. Epub 2017 Dec 2.

Department of Infectious and Tropical Diseases, Jagiellonian University Medical College, Krakow, Poland.

Purpose: There is large variation in the clinical manifestations of Clostridium difficile infection (CDI). We also still can not predict which patients are more susceptible to reinfection with CDI. The aim of our study was to evaluate the effect of gene single nucleotide polymorphisms (SNP) of proinflammatory cytokines, specifically IL-1β, IL-8 on the development, clinical course and recurrence of CDI.

Methods: We performed a prospective study of adults (130 people ≥ 18 years) including 65 patients with CDI treated in tertiary hospital and 65 healthy persons. The following 3 variants were analyzed for the occurrence of gene polymorphisms in patients with CDI versus the control group: IL-1β +3953 A/G (rs1143634), IL-1β -31 A/G (rs1143627), and IL-8 +781 T/C (rs2227306). Then, we assessed the correlation between these genetic polymorphisms and biochemical parameters important in CDI course, CDI severity as well as CDI recurrence.

Results And Conclusions: The presence of genetic polymorphisms of IL-1β +3953 A/G, -31 A/G and IL-8 +781 T/C did not have an effect on the development or recurrence of CDI. The presence of IL-8 +781 T/C polymorphism is associated with the severe CDI.
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http://dx.doi.org/10.1016/j.micpath.2017.11.066DOI Listing
January 2018

Fibrin Clot Permeability as a Predictor of Stroke and Bleeding in Anticoagulated Patients With Atrial Fibrillation.

Stroke 2017 10 13;48(10):2716-2722. Epub 2017 Sep 13.

From the John Paul II Hospital, Krakow, Poland (L.D., A.U.), Centre for Medical Genomics-OMICRON (P.W.), Department of Pharmacology (L.D., P.W.) and Institute of Cardiology (A.U), Jagiellonian University Medical College, Krakow, Poland.

Background And Purpose: Formation of denser fiber networks has been reported in atrial fibrillation and ischemic stroke. In this longitudinal cohort study, we evaluated whether fibrin clot density may predict thromboembolic and bleeding risk in patients with atrial fibrillation on vitamin K antagonists.

Methods: In 236 patients with atrial fibrillation receiving vitamin K antagonists treatment, we measured ex vivo plasma clot permeability (K), a measure of the pore size in fibrin networks.

Results: During a median follow-up of 4.3 (interquartile range, 3.7-4.8) years, annual rates of ischemic stroke or transient ischemic attack and major bleeds were 2.96% and 3.45%, respectively. In multivariate Cox regression analysis, patients with lower K (<6.8 cm×10, median) had increased risk of ischemic stroke or transient ischemic attack (hazard ratio [HR], 6.55; 95% confidence interval [CI], 2.17-19.82) and major bleeds (HR, 10.65; 95% CI, 3.52-32.22). Patients with elevated K (≥6.8 cm×10) had an increased rate of minor bleeding compared with the remainder (11.63% per year versus 3.55% per year; <0.0001). The independent predictors of stroke or transient ischemic attack were low K (<6.8 cm×10; HR, 7.24; 95% CI, 2.53-20.76), age (HR, 1.05; 95% CI, 1.01-1.10), and treatment with angiotensin-converting enzyme inhibitors (HR, 2.27; 95% CI, 1.08-4.77). Major bleeds were predicted by low K (<6.8 cm×10; HR, 8.48; 95% CI, 2.99-24.1) and HAS-BLED score ≥3 (HR, 2.22; 95% CI, 1.12-4.38).

Conclusions: This study is the first to show that unfavorable fibrin properties reflected by formation of denser fibrin networks determine, in part, the efficacy and safety of anticoagulation with vitamin K antagonists in patients with atrial fibrillation.
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http://dx.doi.org/10.1161/STROKEAHA.117.018143DOI Listing
October 2017

Right ventricular morphology and function is not related with microRNAs and fibrosis markers in dilated cardiomyopathy.

Cardiol J 2018 25;25(6):722-731. Epub 2017 Aug 25.

Department of Cardiac and Vascular Diseases, John Paul II Hospital.

Background: The relationship between right ventricle (RV), extracellular matrix (ECM) fibrosis and fibrosis-linked, circulating microRNAs in dilated cardiomyopathy (DCM) is unknown.

Aim: The aim of the study was to uncover the associations between serum markers of ECM metabolism and circulating microRNAs with RV morphological and functional parameters.

Methods: The study population consisted of 70 consecutive DCM patients (ejection fraction 24.4 ± ± 7.4%). Based on detailed echocardiographic assessment - 15 patients had normal RV, whereas 55 patients had RV dilatation (RVD) and/or RV systolic dysfunction (RVSD). Procollagens type I and III carboxy- and amino-terminal peptides, osteopontin (OPN), TGF1-b1, connective tissue growth factor (CTGF), MMP-2, MMP-9 and TIMP-1 were measured in serum as well as expression of miR-21, miR-26, miR-29, miR-30 and miR-133a. All patients underwent endomyocardial biopsy.

Results: Biopsy-proven fibrosis was evenly distributed in two groups. Serum levels of fibrosis markers did not differ between groups. OPN, CTGF, MMP-2, and TIMP-1 correlated with RV parameters. Only miR-133 a was differently expressed in both groups. MiR-21, miR-26, miR-30, and miR-133a cor-related with RV morphological but without functional parameters. Not a single marker of fibrosis was independently associated with RV. MiR-30 was associated with RV impairment in the logistic regression model adjusted for age, sex, body mass index, and disease duration; however, lost its significance in the more comprehensive model.

Conclusions: Right ventricle structural and functional abnormalities are common in DCM. ECM fibrosis and serum markers are not associated with RV impairment. The prognostic value of studied microRNAs on RV is limited in DCM.
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http://dx.doi.org/10.5603/CJ.a2017.0099DOI Listing
June 2019

Vascular transcriptome profiling identifies Sphingosine kinase 1 as a modulator of angiotensin II-induced vascular dysfunction.

Sci Rep 2017 03 9;7:44131. Epub 2017 Mar 9.

Department of Internal and Agricultural Medicine, Faculty of Medicine, Jagiellonian University Medical College, Kraków, Poland.

Vascular dysfunction is an important phenomenon in hypertension. We hypothesized that angiotensin II (AngII) affects transcriptome in the vasculature in a region-specific manner, which may help to identify genes related to vascular dysfunction in AngII-induced hypertension. Mesenteric artery and aortic transcriptome was profiled using Illumina WG-6v2.0 chip in control and AngII infused (490 ng/kg/min) hypertensive mice. Gene set enrichment and leading edge analyses identified Sphingosine kinase 1 (Sphk1) in the highest number of pathways affected by AngII. Sphk1 mRNA, protein and activity were up-regulated in the hypertensive vasculature. Chronic sphingosine-1-phosphate (S1P) infusion resulted in a development of significantly increased vasoconstriction and endothelial dysfunction. AngII-induced hypertension was blunted in Sphk1 mice (systolic BP 167 ± 4.2 vs. 180 ± 3.3 mmHg, p < 0.05), which was associated with decreased aortic and mesenteric vasoconstriction in hypertensive Sphk1 mice. Pharmacological inhibition of S1P synthesis reduced vasoconstriction of mesenteric arteries. While Sphk1 is important in mediating vasoconstriction in hypertension, Sphk1 mice were characterized by enhanced endothelial dysfunction, suggesting a local protective role of Sphk1 in the endothelium. S1P serum level in humans was correlated with endothelial function (arterial tonometry). Thus, vascular transcriptome analysis shows that S1P pathway is critical in the regulation of vascular function in AngII-induced hypertension, although Sphk1 may have opposing roles in the regulation of vasoconstriction and endothelium-dependent vasorelaxation.
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http://dx.doi.org/10.1038/srep44131DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5343497PMC
March 2017

Analysis of gene expression to predict dynamics of future hypertension incidence in type 2 diabetic patients.

BMC Proc 2016 18;10(Suppl 7):113-117. Epub 2016 Oct 18.

Center for Medical Genomics-OMICRON, Jagiellonian University Medical College, Kraków, Poland.

Background: The main focus of the Genetic Analysis Workshop 19 (GAW19) is identification of genes related to the occurrence of hypertension in the cohort of patients with type 2 diabetes mellitus (T2DM). The aim of our study was to predict dynamics of the future hypertension incidence, based on gene expression profiles, systolic and diastolic blood pressure changes in time, sex, baseline age, and cigarette smoking status. We analyzed data made available to GAW19 participants, which included gene expression profiles of peripheral blood mononuclear cells (PBMCs) from the diabetic members of 20 Mexican American families.

Methods: On the basis of mid blood pressure measurements at several time points, the coefficient of regression (slope) was calculated for each individual. We corrected the slope value in patients treated with antihypertensive medications. Feature preprocessing methods were used to remove highly correlated probes and linear dependencies between them. Subsequently, multiple linear regression model was used to associate gene expression with the regression coefficient calculated for each T2DM patient. Tenfold cross-validation was used to validate the model. We used linear mixed effects model and kinship coefficients to account for the family structure. All calculations were performed in R.

Results: This analysis allowed us to identify 6 well-annotated genes: and associated with dynamics of future hypertension incidence. Two of them, and were previously implicated in pathogenesis of hypertension.

Conclusions: There is no obvious mechanism that links all detected genes with dynamics of hypertension incidence. Identification of possible connection with hypertension needs further investigation.
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http://dx.doi.org/10.1186/s12919-016-0015-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5133526PMC
October 2016

Qualitative Parameters of the Colonic Flora in Patients with HNF1A-MODY Are Different from Those Observed in Type 2 Diabetes Mellitus.

J Diabetes Res 2016 11;2016:3876764. Epub 2016 Oct 11.

Department of Metabolic Diseases, Jagiellonian University Medical College, 15 Kopernika Street, 31-501 Kraków, Poland; University Hospital, 36 Kopernika Street, 31-501 Kraków, Poland.

. Type 2 diabetes mellitus (T2DM) is determined by genetic and environmental factors. There have been many studies on the relationship between the composition of the gastrointestinal bacterial flora, T2DM, and obesity. There are no data, however, on the gut microbiome structure in monogenic forms of the disease including Maturity Onset Diabetes of the Young (MODY). . The aim of the investigation was to compare the qualitative parameters of the colonic flora in patients with HNF1AMODY and T2DM and healthy individuals. 16S sequencing of bacterial DNA isolated from the collected fecal samples using the MiSeq platform was performed. . There were significant between-group differences in the bacterial profile. At the phylum level, the amount of Proteobacteria was higher ( = 0.0006) and the amount of Bacteroidetes was lower ( = 0.0005) in T2DM group in comparison to the control group. In HNF1A-MODY group, the frequency of Bacteroidetes was lower than in the control group ( = 0.0143). At the order level, Turicibacterales was more abundant in HNF1A-MODY group than in T2DM group. . It appears that there are differences in the gut microbiome composition between patients with HNF1A-MODY and type 2 diabetes. Further investigation on this matter should be conducted.
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http://dx.doi.org/10.1155/2016/3876764DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5078663PMC
June 2017

Resolvin D1 down-regulates CYP1A1 and PTGS2 gene in the HUVEC cells treated with benzo(a)pyrene.

Pharmacol Rep 2016 Oct 27;68(5):939-44. Epub 2016 Jun 27.

Department of Medicine, University of Connecticut Health Center, Farmington, CT, USA.

Background: Polycyclic aromatic hydrocarbons (PAHs) can interact with lipids and their derivatives and have been known to induce atherosclerosis. The aim of this study was to evaluate the impact of Resolvin D1 (RvD1) on inflammatory-state realted proteins and genes in the human primary umbilical vein endothelial HUVEC cells exposed to benzo(a)pyrene (BaP).

Methods: We analyzed the influence of RvD1 and/or BaP on cyclooxygenase-2 (COX-2), cytosolic prostaglandine E2 synthase (cPGES), glutathione S transferase (GSTM1) and aryl hydrocarbon receptor (AhR) protein expression by Western blot. Additionaly, phospholipase A2 (cPLA2) and cytochrome P450 (CYP1A1) activity, as well as AhR, CYP1A1, phospholipase A2 (PLA2G4A) and prostaglandin synthase 2 (PTGS2) gene expression by qRT-PCR was studied.

Results: RvD1 down-regulates cytochrome P450 (CYP1A1) and prostaglandin synthase 2 (PTGS2) gene expression in HUVEC cells exposed to BaP. Repressesion of COX-2, cPGES and overexpressesion of GSTM1 protein was noted after co-treatment with RvD1 and BaP. After incubation with RvD1 an increase of cPLA2 and a decrease of CYP1A1 activity was observed when compared to BaP treated alone endothelial cells.

Conclusions: Our data suggests that RvD1 can significantly contributes on vascular function and alleviates the harmful effects caused by BaP, which might potentially aid in the repair of the injured endothelium.
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http://dx.doi.org/10.1016/j.pharep.2016.05.005DOI Listing
October 2016