MicroRNA Profiling in Papillary Thyroid Cancer.

Genetic alterations are well known to be related to the pathogenesis and prognosis of papillary thyroid carcinoma (PTC). Some miRNA expression dysregulations have previously been described in the context of cancer development including thyroid carcinoma. In our study, we performed original molecular diagnostics on tissue samples related to our own patients. We aimed to identify all dysregulated miRNAs in potential association with PTC development via sequencing much higher numbers of control-matched PTC tissue samples and analyzing a wider variety of miRNA types than previous studies. We analyzed the expression levels of 2656 different human miRNAs in the context of 236 thyroid tissue samples (118 tumor and control pairs) related to anonymized PTC cases. Also, KEGG pathway enrichment analysis and GO framework analysis were used to establish the links between miRNA dysregulation and certain biological processes, pathways of signaling, molecular functions, and cellular components. A total of 30 significant differential miRNA expressions with at least ±1 log2 fold change were found related to PTC including, e.g., miR-551b, miR-146b, miR-221, miR-222, and miR-375, among others, being highly upregulated, as well as miR-873 and miR-204 being downregulated. In addition, we identified miRNA patterns in vast databases (KEGG and GO) closely similar to that of PTC including, e.g., miRNA patterns of prostate cancer, HTLV infection, HIF-1 signaling, cellular responses to growth factor stimulus and organic substance, and negative regulation of gene expression. We also found 352 potential associations between certain miRNA expressions and states of clinicopathological variables. Our findings-supported by the largest case number of original matched-control PTC-miRNA relation research-suggest a distinct miRNA expression profile in PTC that could contribute to a deeper understanding of the underlying molecular mechanisms promoting the pathogenesis of the disease. Moreover, significant miRNA expression deviations and their signaling pathways in PTC presented in our study may serve as potential biomarkers for PTC diagnosis and prognosis or even therapeutic targets in the future.

Procalcitonin-guided antibiotic therapy may shorten length of treatment and may improve survival-a systematic review and meta-analysis.

BACKGROUND: Appropriate antibiotic (AB) therapy remains a challenge in the intensive care unit (ICU). Procalcitonin (PCT)-guided AB stewardship could help optimize AB treatment and decrease AB-related adverse effects, but firm evidence is still lacking. Our aim was to compare the effects of PCT-guided AB therapy with standard of care (SOC) in critically ill patients. METHODS: We searched databases CENTRAL, Embase and Medline. We included randomized controlled trials (RCTs) comparing PCT-guided AB therapy (PCT group) with SOC reporting on length of AB therapy, mortality, recurrent and secondary infection, ICU length of stay (LOS), hospital LOS or healthcare costs. Due to recent changes in sepsis definitions, subgroup analyses were performed in studies applying the Sepsis-3 definition. In the statistical analysis, a random-effects model was used to pool effect sizes. RESULTS: We included 26 RCTs (n = 9048 patients) in the quantitative analysis. In comparison with SOC, length of AB therapy was significantly shorter in the PCT group (MD - 1.79 days, 95% CI: -2.65, - 0.92) and was associated with a significantly lower 28-day mortality (OR 0.84, 95% CI: 0.74, 0.95). In Sepsis-3 patients, mortality benefit was more pronounced (OR 0.46 95% CI: 0.27, 0.79). Odds of recurrent infection were significantly higher in the PCT group (OR 1.36, 95% CI: 1.10, 1.68), but there was no significant difference in the odds of secondary infection (OR 0.81, 95% CI: 0.54, 1.21), ICU and hospital length of stay (MD - 0.67 days 95% CI: - 1.76, 0.41 and MD - 1.23 days, 95% CI: - 3.13, 0.67, respectively). CONCLUSIONS: PCT-guided AB therapy may be associated with reduced AB use, lower 28-day mortality but higher infection recurrence, with similar ICU and hospital length of stay. Our results render the need for better designed studies investigating the role of PCT-guided AB stewardship in critically ill patients.

A biallelic multiple nucleotide length polymorphism explains functional causality at 5p15.33 prostate cancer risk locus.

To date, single-nucleotide polymorphisms (SNPs) have been the most intensively investigated class of polymorphisms in genome wide associations studies (GWAS), however, other classes such as insertion-deletion or multiple nucleotide length polymorphism (MNLPs) may also confer disease risk. Multiple reports have shown that the 5p15.33 prostate cancer risk region is a particularly strong expression quantitative trait locus (eQTL) for Iroquois Homeobox 4 (IRX4) transcripts. Here, we demonstrate using epigenome and genome editing that a biallelic (21 and 47 base pairs (bp)) MNLP is the causal variant regulating IRX4 transcript levels. In LNCaP prostate cancer cells (homozygous for the 21 bp short allele), a single copy knock-in of the 47 bp long allele potently alters the chromatin state, enabling de novo functional binding of the androgen receptor (AR) associated with increased chromatin accessibility, Histone 3 lysine 27 acetylation (H3K27ac), and ~3-fold upregulation of IRX4 expression. We further show that an MNLP is amongst the strongest candidate susceptibility variants at two additional prostate cancer risk loci. We estimated that at least 5% of prostate cancer risk loci could be explained by functional non-SNP causal variants, which may have broader implications for other cancers GWAS. More generally, our results underscore the importance of investigating other classes of inherited variation as causal mediators of human traits.

Methodological and Biological Factors Influencing Global DNA Methylation Results Measured by LINE-1 Pyrosequencing Assay in Colorectal Tissue and Liquid Biopsy Samples.

Long interspersed nuclear element 1 (LINE-1) bisulfite pyrosequencing is a widely used technique for genome-wide methylation analyses. We aimed to investigate the effects of experimental and biological factors on its results to improve the comparability. LINE-1 bisulfite pyrosequencing was performed on colorectal tissue (n = 222), buffy coat (n = 39), and plasma samples (n = 9) of healthy individuals and patients with colorectal tumors. Significantly altered methylation was observed between investigated LINE-1 CpG positions of non-tumorous tissues (p ≤ 0.01). Formalin-fixed, paraffin-embedded biopsies (73.0 ± 5.3%) resulted in lower methylation than fresh frozen samples (76.1 ± 2.8%) (p ≤ 0.01). DNA specimens after long-term storage showed higher methylation levels (+3.2%, p ≤ 0.01). In blood collection tubes with preservatives, cfDNA and buffy coat methylation significantly changed compared to K3EDTA tubes (p ≤ 0.05). Lower methylation was detected in older (>40 years, 76.8 ± 1.7%) vs. younger (78.1 ± 1.0%) female patients (p ≤ 0.05), and also in adenomatous tissues with MTHFR 677CT, or 1298AC mutations vs. wild-type (p ≤ 0.05) comparisons. Based on our findings, it is highly recommended to consider the application of standard DNA samples in the case of a possible clinical screening approach, as well as in experimental research studies.

Global DNA hypomethylation of colorectal tumours detected in tissue and liquid biopsies may be related to decreased methyl-donor content.

BACKGROUND: Hypomethylation of long interspersed nuclear element 1 (LINE-1) is characteristic of various cancer types, including colorectal cancer (CRC). Malfunction of several factors or alteration of methyl-donor molecules' (folic acid and S-adenosylmethionine) availability can contribute to DNA methylation changes. Detection of epigenetic alterations in liquid biopsies can assist in the early recognition of CRC. Following the investigations of a Hungarian colon tissue sample set, our goal was to examine the LINE-1 methylation of blood samples along the colorectal adenoma-carcinoma sequence and in inflammatory bowel disease. Moreover, we aimed to explore the possible underlying mechanisms of global DNA hypomethylation formation on a multi-level aspect. METHODS: LINE-1 methylation of colon tissue (n = 183) and plasma (n = 48) samples of healthy controls and patients with colorectal tumours were examined with bisulfite pyrosequencing. To investigate mRNA expression, microarray analysis results were reanalysed in silico (n = 60). Immunohistochemistry staining was used to validate DNA methyltransferases (DNMTs) and folate receptor beta (FOLR2) expression along with the determination of methyl-donor molecules' in situ level (n = 40). RESULTS: Significantly decreased LINE-1 methylation level was observed in line with cancer progression both in tissue (adenoma: 72.7 ± 4.8%, and CRC: 69.7 ± 7.6% vs. normal: 77.5 ± 1.7%, p ≤ 0.01) and liquid biopsies (adenoma: 80.0 ± 1.7%, and CRC: 79.8 ± 1.3% vs. normal: 82.0 ± 2.0%, p ≤ 0.01). However, no significant changes were recognized in inflammatory bowel disease cases. According to in silico analysis of microarray data, altered mRNA levels of several DNA methylation-related enzymes were detected in tumours vs. healthy biopsies, namely one-carbon metabolism-related genes-which met our analysing criteria-showed upregulation, while FOLR2 was downregulated. Using immunohistochemistry, DNMTs, and FOLR2 expression were confirmed. Moreover, significantly diminished folic acid and S-adenosylmethionine levels were observed in parallel with decreasing 5-methylcytosine staining in tumours compared to normal adjacent to tumour tissues (p ≤ 0.05). CONCLUSION: Our results suggest that LINE-1 hypomethylation may have a distinguishing value in precancerous stages compared to healthy samples in liquid biopsies. Furthermore, the reduction of global DNA methylation level could be linked to reduced methyl-donor availability with the contribution of decreased FOLR2 expression.

Two Sides to Every Question: Attempts to Activate Chicken Innate Immunity in 2D and 3D Hepatic Cell Cultures.

The liver with resident tissue macrophages is the site of vivid innate immunity, activated also by pathogen-associated molecular patterns (PAMPs) leaking through the intestinal barrier. As gut-derived inflammatory diseases are of outstanding importance in broiler chickens, the present study aimed to establish a proper hepatic inflammatory model by comparing the action of different PAMPs from poultry pathogens on chicken 2D and 3D primary hepatocyte-non-parenchymal cell co-cultures, the latter newly developed with a magnetic bioprinting method. The cultures were challenged by the bacterial endotoxins lipopolysaccharide (LPS) from Escherichia coli, lipoteichoic acid (LTA) from Staphylococcus aureus and by enterotoxin (ETxB) from Escherichia coli, Salmonella Typhimurium derived flagellin, phorbol myristate acetate (PMA) as a model proinflammatory agent and polyinosinic polycytidylic acid (poly I:C) for mimicking viral RNA exposure. Cellular metabolic activity was assessed with the CCK-8 test, membrane damage was monitored with the lactate dehydrogenase (LDH) leakage assay and interleukin-6 and -8 (Il-6 and -8) concentrations were measured in cell culture medium with a chicken specific ELISA. Both LPS and LTA increased the metabolic activity of the 3D cultures, concomitantly decreasing the LDH leakage, while in 2D cultures ETxB stimulated, PMA and poly I:C depressed the metabolic activity. Based on the moderately increased extracellular LDH activity, LTA seemed to diminish cell membrane integrity in 2D and poly I:C in both cell culture models. The applied endotoxins remarkably reduced the IL-8 release of 3D cultured cells, suggesting the effective metabolic adaptation and the presumably initiated anti-inflammatory mechanisms of the 3D spheroids. Notwithstanding that the IL-6 and IL-8 production of 2D cells was mostly not influenced by the endotoxins used, only the higher LTA dose was capable to evoke an IL-8 surge. Flagellin, PMA and poly I:C exerted proinflammatory action in certain concentrations in both 2D and 3D cultures, reflected by the increased cellular IL-6 release. Based on these data, LTA, flagellin, PMA and poly I:C can be considered as potent candidates to induce inflammation in chicken primary hepatic cell cultures, while LPS failed to trigger proinflammatory cytokine production, suggesting the relatively high tolerance of avian liver cells to certain bacterial endotoxins. These results substantiate that the established 3D co-cultures seemed to be proper tools for testing potential proinflammatory molecules; however, the remarkable differences between 2D and 3D models should be addressed and further studied.

Hybrid robotic versus hybrid laparoscopic Ivor Lewis oesophagectomy: a case-matched analysis.

OBJECTIVES: Robotic-assisted oesophagectomy for cancer has been increasingly employed worldwide; however, the benefits of this technique compared to conventional minimally invasive oesophagectomy are unclear. Since 2016, hybrid robotic minimally invasive oesophagectomy (R-HMIE) has increasingly replaced hybrid laparoscopic minimally invasive oesophagectomy (HMIE) as the standard of care in our institution. The aim of this study was to compare these procedures. METHODS: Over a 10-year period, 686 patients underwent oesophagectomy at our institution. Out of these patients, 128 patients with cancer were treated with a hybrid minimally invasive technique. Each patient who underwent R-HMIE was matched according to gender, age, comorbidity, American Society of Anesthesiologists classification, Union International Contre le Cancer stage, localization, histology and neoadjuvant treatment with a patient who underwent HMIE. Perioperative parameters were extracted from our database and compared between the 2 groups. RESULTS: After the matching procedure, 88 patients were included in the study. Between HMIE and R-HMIE, no significant differences (P > 0.05) were found in operating time (median 281 vs 300 min), R0 resection rate (n = 42 vs 42), harvested lymph nodes (median 28 vs 24), hospital stay (median 19 vs 17 days) and intensive care unit stay (median 7 vs 6.5 days). Regarding surgical complications, no difference could be observed either (n = 42 vs 44). CONCLUSIONS: Minimally invasive oesophagectomy remains a challenging operation with high morbidity even in a high-volume institution. According to our intra- and short-term results, we have found no difference between R-HMIE and HMIE.