Intratumor heterogeneity in colorectal cancer: Distribution of tumor suppressor gene variants with regard to patient lymph node status.

Intratumor heterogeneity (ITH) results from accumulation of somatic mutations in the fractions of successive cancer cell generations. We aimed to use deep sequencing to investigate ITH in colorectal tumors with particular emphasis on variants in oncogenes (ONC) and tumor suppressor genes (TSG). Samples were collected from 16 patients with colorectal cancer and negative or positive lymph node status (n = 8 each). We deep-sequenced a panel of 56 cancer-related genes in the central and peripheral locations of T3 size primary tumors and healthy mucosa. The central region of T3 tumors has a different frequency profile and composition of genetic variants. This mutation profile is capable of independently discriminating patients with different lymph node status (p = 0.028) in the central region. We noted an increasing number of mutations outside of the central region of the tumor and a higher number of mutations in tumors from node-positive patients. Unexpectedly, in the healthy mucosa, we identified somatic mutations with variant allele frequencies, characteristic not only of heterozygotes and homozygotes but also of other discrete peaks (e.g., around 10%, 20%), suggestive of clonal expansion of certain mutant alleles. We found differences in the distribution of variant allele frequencies in TSGs when comparing node-negative and node-positive tumors (p = 0.029), as well as central and peripheral regions (p = 0.00399). TSGs may play an important role in the escape of the tumor toward metastatic colonization.

Expression of micro-ribonucleic acids in thyroid nodules and serum to discriminate between follicular adenoma and cancer in patients with a fine needle aspiration biopsy classified as suspicious for follicular neoplasm: A preliminary study.

BACKGROUND: Approximately 10% of thyroid nodules undergoing fine needle aspiration biopsy (FNAB) receive a suspicious for follicular neoplasm (SFN) classification. Currently, there is no diagnostic tool to preoperatively discriminate between follicular adenoma (FA) and thyroid cancer (TC), and most patients require surgery to exclude malignancy. OBJECTIVES: To characterize the micro-ribonucleic acid (miRNA) signature of tumors assessed as SFN and define circulating miRNA patterns to distinguish FA from follicular cancer in patients with thyroid nodules biopsied using FNAB. MATERIAL AND METHODS: The study included excised tumor and thyroid tissue samples from 80 consecutive patients collected by a pathologist in the operating theater. The miRNA was isolated from specimens at the Center for Medical Genomics OMICRON, and next-generation sequencing (NGS) was used to obtain target miRNAs. In addition, miRNA expression was detected in serum using polymerase chain reaction (PCR). RESULTS: Well-differentiated thyroid cancer (WDTC) samples had significantly higher expression levels of hsa-miR-146b-5p (p = 0.030) and hsa-miR-146b-3p (p = 0.032), while the expression levels of hsa-miR-195-3p were significantly lower (p = 0.032) in WDTC samples compared to FA specimens. The serum of TC patients showed markedly higher expression of the unique miRNA hsa-miR-195-3p (p = 0.039). CONCLUSIONS: The overexpression of hsa-miR-146b-5p and hsa-miR-146b-3p, and the downregulation of hsa-miR-195-3p expression could be used as biomarkers to distinguish FA from WDTC in patients with FNAB results classified as Bethesda tier IV. In addition, hsa-miR-195-3p could act as a serum biomarker for differentiating patients with FA from those with WDTC, and preoperative measurement of its expression would help avoid unnecessary surgeries. However, this concept needs further verification in a more substantial prospective study.

MicroRNA composition of plasma extracellular vesicles: a harbinger of late cardiotoxicity of doxorubicin.

BACKGROUND: The use of doxorubicin is associated with an increased risk of acute and long-term cardiomyopathy. Despite the constantly growing number of cancer survivors, little is known about the transcriptional mechanisms which progress in the time leading to a severe cardiac outcome. It is also unclear whether long-term transcriptomic alterations related to doxorubicin use are similar to transcriptomic patterns present in patients suffering from other cardiomyopathies. METHODS: We have sequenced miRNA from total plasma and extracellular vesicles (EVs) from 66 acute lymphoblastic leukemia (ALL) survivors and 61 healthy controls (254 samples in total). We then analyzed processes regulated by differentially expressed circulating miRNAs and cross-validated results with the data of patients with clinically manifested cardiomyopathies. RESULTS: We found that especially miRNAs contained within EVs may be informative in terms of cardiomyopathy development and may regulate pathways related to neurotrophin signaling, transforming growth factor beta (TGFß) or epidermal growth factor receptors (ErbB). We identified vesicular miR-144-3p and miR-423-3p as the most variable between groups and significantly correlated with echocardiographic parameters and, respectively, for plasma: let-7g-5p and miR-16-2-3p. Moreover, vesicular miR-144-3p correlates with the highest number of echocardiographic parameters and is differentially expressed in the circulation of patients with dilated cardiomyopathy. We also found that distribution of particular miRNAs between of plasma and EVs (proportion between compartments) e.g., miR-184 in ALL, is altered, suggesting changes within secretory and miRNA sorting mechanisms. CONCLUSIONS: Our results show that transcriptomic changes resulting from doxorubicin induced myocardial injury are reflected in circulating miRNA levels and precede development of the late onset cardiomyopathy phenotype. Among miRNAs related to cardiac function, we found vesicular miR-144-3p and miR-423-3p, as well as let-7g-5p and miR-16-2-3p contained in the total plasma. Selection of source for such studies (plasma or EVs) is of critical importance, as distribution of some miRNA between plasma and EVs is altered in ALL survivors, in comparison to healthy people, which suggests that doxorubicin-induced changes include miRNA sorting and export to extracellular space.

Variants influencing age at diagnosis of HNF1A-MODY.

BACKGROUND: HNF1A-MODY is a monogenic form of diabetes caused by variants in the HNF1A gene. Different HNF1A variants are associated with differences in age of disease onset, but other factors are postulated to influence this trait. Here, we searched for genetic variants influencing age of HNF1A-MODY onset. METHODS: Blood samples from 843 HNF1A-MODY patients from Czech Republic, France, Poland, Slovakia, the UK and the US were collected. A validation set consisted of 121 patients from the US. We conducted a genome-wide association study in 843 HNF1A-MODY patients. Samples were genotyped using Illumina Human Core arrays. The core analysis was performed using the GENESIS package in R statistical software. Kinship coefficients were estimated with the KING and PC-Relate algorithms. In the linear mixed model, we accounted for year of birth, sex, and location of the HNF1A causative variant. RESULTS: A suggestive association with age of disease onset was observed for rs2305198 (p = 2.09E-07) and rs7079157 (p = 3.96E-06) in the HK1 gene, rs2637248 in the LRMDA gene (p = 2.44E-05), and intergenic variant rs2825115 (p = 2.04E-05). Variant rs2637248 reached nominal significance (p = 0.019), while rs7079157 (p = 0.058) and rs2825115 (p = 0.068) showed suggestive association with age at diabetes onset in the validation set. CONCLUSIONS: rs2637248 in the LRMDA gene is associated with age at diabetes onset in HNF1A-MODY patients.

Mutation search within monogenic diabetes genes in Polish patients with long-term type 1 diabetes and preserved kidney function.

INTRODUCTION: Some patients with type 1 diabetes (T1DM) are free from advanced complications despite long­standing disease. These patients may be carriers of gene mutations responsible for maturity­onset diabetes of the young and may have been misdiagnosed with T1DM. OBJECTIVES: We aimed to determine the clinical characteristics of patients with long­term T1DM, without advanced microvascular complications, and with well­preserved kidney function. A search for mutations in monogenic diabetes genes was performed. PATIENTS AND METHODS: Patients were recruited at 2 Polish university centers based on the following criteria: T1DM duration of 40 years or longer and absence of advanced complications defined as chronic kidney disease (estimated glomerular filtration rate [eGFR] <60 ml/min/1.73 m2 ), overt proteinuria, blindness, and diabetic foot syndrome. Mutations in the 7 most frequent monogenic diabetes genes were identified using next­generation sequencing. RESULTS: We enrolled 45 patients with T1DM (mean [SD] age at examination, 59.2 [8.0] years; mean [SD] age at T1DM diagnosis, 14.6 [6.7] years). Mean (SD) hemoglobin A1c levels were 7.6% (1.4%); daily insulin dose, 0.48 (0.17) U/kg; high­density lipoprotein (HDL) cholesterol levels, 1.9 (0.6) mmol/l; body mass index (BMI), 26.4 (5.0) kg/m2 ; and eGFR, 82.2 (12.1) ml/min/1.73 m2 . Albuminuria and retinopathy were reported in 7 and 39 patients, respectively. We were not able to assign a causative role to any of 10 genetic variants identified by next­generation sequencing in this cohort. CONCLUSIONS: Patients with long­term T1DM and preserved kidney function have good glycemic control, elevated HDL cholesterol levels, low insulin requirements, near ­normal BMI, and a rare occurrence of mutations in monogenic diabetes genes.

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

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 (n = 10), 2 (n = 7), and 3 (n = 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.

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

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 (CHEK2) and TP53 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 CHEK2 gene amongst 5 different variants, and the intron variant c.319+379A>G was the most common SNP. In the TP53 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 CHEK2 and TP53 genes.

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

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 (p = 0.02), and Shannon's (p = 0.04) and Simpson's diversity indexes (p = 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 (Ruminococcaceae) was enriched in patients with HbA1c < 53 mmol/mol, whereas one family (Streptococcaceae) and four species (Ruminococcus torques, unclassified species of Lactococcus, Eubacteroim dolichum, and Coprobacillus cateniformis) 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.

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

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 < 10-4) 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 < 10-4) 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.

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

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 Lactobacillus 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 Lactobacillus iners, Lactobacillus gasseri, and Lactobacillusplantarum. Statistically significant differences in the quantitative composition between trimesters were observed in relation to Lactobacillus jensenii,Streptococcus agalactiae, Lactobacillus iners, Gardnerella spp. Out of the 32 patients, 20 demonstrated fluctuations within the genus Lactobacillus, and 9 of them, at different stages of pregnancy, exhibited the presence of potentially pathogenic microbiota, among others: Streptococcus agalactiae, Gardnerella spp., Atopobium vaginae, and Enterococcus faecalis. 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.

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

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 FER1L4 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.

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

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 (STK11, RET, FANCM, SLX4, WRN, MEN1, and KIT) 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 (RET), rs10138997 (FANCM), rs2230009 (WRN), and rs2959656 (MEN1). 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: SDHB (rs2746462), ALK (rs1670283), XPC (rs2958057), RECQL4 (rs4925828; rs11342077, rs398010167; rs2721190), DDB2 (rs326212), MEN1 (rs540012), AIP (rs4930199), ATM (rs659243), HNF1A (rs1169305), BRCA2 (rs206075; rs169547), ERCC5 (rs9514066; rs9514067), and FANCI (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.

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

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 (ß = - 0.69, p = 0.056). This BMI association was confirmed on 1889 patients from FHS cohort (ß = - 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.

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

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 cm2 vs. 1.1 cm2 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.

Effects of rivaroxaban and dabigatran on local expression of coagulation and inflammatory factors within human aortic stenotic valves.

BACKGROUND: Treatment with non-vitamin K antagonist oral anticoagulants (NOACs) such as dabigatran (a direct thrombin inhibitor) or rivaroxaban (a direct inhibitor of factor [F] Xa) attenuates atherosclerotic plaque progression in hypercholesterolemic mice. PURPOSE: To evaluate the effect of NOACs application on the expression of coagulation proteins in loco within stenotic aortic valves and in valve interstitial cells (VICs) from patients with severe aortic stenosis (AS). METHODS: Primary cultures of VICs obtained from 90 patients undergoing aortic valve replacement were stimulated with TNF-α (50 ng/mL) and pre-treated with rivaroxaban (1 and 10 ng/mL) or dabigatran (25 and 250 ng/mL). The expression of coagulation proteins was analyzed by immunofluorescence. Cytokine levels were measured by ELISA. RESULTS: FX, FXa, FVII, thrombin and PAR1/2 were present in loco within human aortic stenotic valves. Cultured VICs exhibited constant expression of FX, TF, PAR1/2. Exposure of VICs to TNF-α caused the upregulated expression of TF, PAR1/2 and induced expression of thrombin, FVII and FXa. FX was expressed by 80% of VICs, regardless of stimulation. Cultured VICs were able to synthesize metalloproteinases 1-3, IL-6, IL-32, IL-34, osteopontin and osteocalcin, the levels of which increased under TNF-α stimulation. NOACs added to culture inhibited coagulation factor and PAR1/2 expression. Moreover, NOACs down-regulated VIC-derived proteins responsible for valve calcification and extracellular matrix remodeling. CONCLUSIONS: NOACs at therapeutic concentrations may inhibit the effects of FXa and thrombin at in vitro level. It might be speculated that long-term treatment with rivaroxaban or dabigatran could attenuate the progression of AS in humans.

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

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 (p = 0.003, adj. p = 0.010) and observed operational taxonomic units (OTUs) (p = 0.007, adj. p = 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.

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

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.

Contribution of a Novel B3GLCT Variant to Peters Plus Syndrome Discovered by a Combination of Next-Generation Sequencing and Automated Text Mining.

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 B3GLCT 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 B3GLCT gene product, O-fucose-specific ß-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 B3GLCT 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.

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

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.

First insight into microbiome profile of fungivorous thrips Hoplothrips carpathicus (Insecta: Thysanoptera) at different developmental stages: molecular evidence of Wolbachia endosymbiosis.

Insects' exoskeleton, gut, hemocoel, and cells are colonized by various microorganisms that often play important roles in their host life. Moreover, insects are frequently infected by vertically transmitted symbionts that can manipulate their reproduction. The aims of this study were the characterization of bacterial communities of four developmental stages of the fungivorous species Hoplothrips carpathicus (Thysanoptera: Phlaeothripidae), verification of the presence of Wolbachia, in silico prediction of metabolic potentials of the microorganisms, and sequencing its mitochondrial COI barcode. Taxonomy-based analysis indicated that the bacterial community of H. carpathicus contained 21 bacterial phyla. The most abundant phyla were Proteobacteria, Actinobacteria, Bacterioidetes and Firmicutes, and the most abundant classes were Alphaproteobacteria, Actinobacteria, Gammaproteobacteria and Betaproteobacteria, with different proportions in the total share. For pupa and imago (adult) the most abundant genus was Wolbachia, which comprised 69.95% and 56.11% of total bacterial population respectively. Moreover, similarity analysis of bacterial communities showed that changes in microbiome composition are congruent with the successive stages of H. carpathicus development. PICRUSt analysis predicted that each bacterial community should be rich in genes involved in membrane transport, amino acid metabolism, carbohydrate metabolism, replication and repair processes.