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BackgroundSince 2000, we have been grafting the right coronary artery system (RCAs) using the proximal portion of the right internal thoracic artery (RITA) as the inflow of the saphenous vein graft (SVG) to increase the number of patients undergoing beating heart complete myocardial revascularization.MethodsFrom 2000 to 2022, 928 consecutive patients underwent SVG on the RCAs. In 546 patients (58.8%), the inflow was the RITA (I-graft group), and in 382 patients (41.2%), the inflow was the aorta (Ao-graft group). The inclusion criteria were age ≤75 years, ejection fraction >35%, only one SVG per patient, bilateral internal thoracic arteries as a Y-graft on the left system (three-vessel disease, n = 817, 88.0%) or left internal thoracic artery on the left anterior descending artery and RITA + SVG on the RCAs (two-vessel disease, n = 111, 12.0%). Propensity matching identified 306 patients per group. After a median follow-up of 8 (5–10) years, graft patency was assessed by coronary computed tomographic angiography in 132 patients (64 in the I-graft group and 68 in the Ao-graft group).ResultsEarly results were similar in both groups. The I-graft group had higher 10-year survival and freedom from main adverse cardiac events (90.0 ± 2.0 vs. 80.6 ± 3.8, p = 0.0162, and 81.3 ± 2.7 vs. 64.7 ± 5.6, p = 0.0206, respectively). When RITA was the inflow, SVG had a higher estimated 10-year patency rate (82.8% ± 6.5 vs. 58.8% ± 7.4, p = 0.0026) and a smaller inner lumen diameter (2.7 ± 0.4 vs. 3.4 ± 0.6 mm, p

Background: Burn injuries remain a major clinical problem worldwide, which require special management by experienced plastic surgeons. However, they cannot be available in every healthcare unit; consequently, there is a need for effective treatment options that could be utilized by a wide range of non-expert healthcare professionals. The aim of the present experimental study was to investigate the safety and efficacy of using a fibrin sealant (TISSEELTM) compared to the conventional treatment with sulfadiazine on partial-thickness burn in a rat animal model. Methods: A cohort of Sprague Dawley rats underwent partial-thickness contact thermal burn wounds and were divided into three study groups: control group (no treatment), silver sulfadiazine cream group and TISSEELTM group. Following animal sacrifice, a blinded histopathologic analysis was conducted regarding inflammatory response, healing and tissue regeneration. Results: In total, 30 animals were included with a median weight of 236 ± 10 g. Two animals from the control group died on the first postoperative day. Animals in the TISSEELTM group presented dominant collagen expression compared to animals in the control and silver sulfadiazine cream group (p = 0.000). Histopathologic analysis also demonstrated marked leukocyte infiltration (p = 0.009), increased neovascularization (p = 0.000) and higher fibroblast expression (p = 0.002) in the TISSEELTM group compared to the other two groups. Conclusions: TISSEELTM seems to be a safe alternative (or even principal) option for the initial therapeutic approach of partial-thickness burn injuries. Moreover, it seems to be superior to silver sulfadiazine in terms of tissue healing and regeneration. However, additional experimental as well as clinical research is necessary prior to implementation in clinical practice.

Background and Aims: Decoding pancreatic ductal adenocarcinoma heterogeneity and the consequent therapeutic selection remains a challenge. We aimed to characterize epigenetically regulated pathways involved in pancreatic ductal adenocarcinoma progression. Methods: Global DNA methylation analysis in pancreatic cancer patient tissues and cell lines was performed to identify differentially methylated genes. Targeted bisulfite sequencing and in vitro methylation reporter assays were employed to investigate the direct link between site-specific methylation and transcriptional regulation. A series of in vitro loss-of-function and gain-of function studies and in vivo xenograft and the KPC (LSL-KrasG12D/+; LSL-Trp53R172H/+; Pdx1-Cre) mouse models were used to assess pancreatic cancer cell properties. Gene and protein expression analyses were performed in 3 different cohorts of pancreatic cancer patients and correlated to clinicopathological parameters. Results: We identify Hepatocyte Nuclear Factor 4A (HNF4A) as a novel target of hypermethylation in pancreatic cancer and demonstrate that site-specific proximal promoter methylation drives HNF4A transcriptional repression. Expression analyses in patients indicate the methylation-associated suppression of HNF4A expression in pancreatic cancer tissues. In vitro and in vivo studies reveal that HNF4A is a novel tumor suppressor in pancreatic cancer, regulating cancer growth and aggressiveness. As evidenced in both the KPC mouse model and human pancreatic cancer tissues, HNF4A expression declines significantly in the early stages of the disease. Most importantly, HNF4 loss correlates with poor overall patient survival. Conclusion: HNF4A silencing, mediated by promoter DNA methylation, drives pancreatic cancer development and aggressiveness leading to poor patient survival.

Background: The widespread use of fluoroquinolones has been associated with the formation, dissection, and rupture of aortic aneurysms. Arterial biomarkers are established predictors of cardiovascular events. The present study was designed to investigate the effect of quinolones on arterial stiffness and aortic size for the first time. Methods: We studied 28 subjects receiving short-term (Results: Subjects had similar values of arterial biomarkers, blood pressure measurements, and aortic diameters at baseline. At follow-up, no significant change was observed between the two groups regarding the hemodynamic parameters and arterial biomarkers (p > 0.05 for all), i.e., cfPWV (7.9 ± 2.6 m/s for the control group vs. 8.1 ± 2.4 m/s for the fluoroquinolones group; p = 0.79), AIx@75 (22.6 ± 9.0% for the control group vs. 26.6 ± 8.1% for the fluoroquinolones group; p = 0.09), and aortic diameters. Conclusions: To our knowledge, FRAGILES is the first study to provide insights into the possible effects of fluoroquinolones on arterial biomarkers, showing that, at least in the short term, treatment with fluoroquinolones does not affect aortic function and diameter.

Background: There is a need for clinical markers to aid in the detection of individuals at risk of harboring an ascending thoracic aneurysm (ATAA) or developing one in the future. Objectives: To our knowledge, ATAA remains without a specific biomarker. This study aims to identify potential biomarkers for ATAA using targeted proteomic analysis. Methods: In this study, 52 patients were divided into three groups depending on their ascending aorta diameter: 4.0–4.5 cm (N = 23), 4.6–5.0 cm (N = 20), and >5.0 cm (N = 9). A total of 30 controls were in-house populations ethnically matched to cases without known or visible ATAA-related symptoms and with no ATAA familial history. Before the debut of our study, all patients provided medical history and underwent physical examination. Diagnosis was confirmed by echocardiography and angio-computed tomography (CT) scans. Targeted-proteomic analysis was conducted to identify possible biomarkers for the diagnosis of ATAA. Results: A Kruskal–Wallis test revealed that C-C motif chemokine ligand 5 (CCL5), defensin beta 1 (HBD1), intracellular adhesion molecule-1 (ICAM1), interleukin-8 (IL8), tumor necrosis factor alpha (TNFα) and transforming growth factor-beta 1 (TGFB1) expressions are significantly increased in ATAA patients in comparison to control subjects with physiological aorta diameter (p < 0.0001). The receiver-operating characteristic analysis showed that the area under the curve values for CCL5 (0.84), HBD1 (0.83) and ICAM1 (0.83) were superior to that of the other analyzed proteins. Conclusions: CCL5, HBD1 and ICAM1 are very promising biomarkers with satisfying sensitivity and specificity that could be helpful in stratifying risk for the development of ATAA. These biomarkers may assist in the diagnosis and follow-up of patients at risk of developing ATAA. This retrospective study is very encouraging; however, further in-depth studies may be worthwhile to investigate the role of these biomarkers in the pathogenesis of ATAA.

Paragangliomas are rare tumors of chromaffin cells arising from an extra-adrenal location. Unlike pheochromocytomas, they are seldom functional. We present a case of pericardial paraganglioma incidentally encountered on an echocardiographic study, focusing on the characteristic features the tumor demonstrates on different imaging modalities. (Level of Difficulty: Intermediate.)

International experts gathered at the Mayo Clinic (Rochester MN, USA) on February 27th-28th, 2017 for a meeting entitled ‘Basic and Translational Facets of the Epigenetics of GI Diseases’. This workshop summarized recent advances on the role of epigenetics in the pathobiology of gastrointestinal (GI) diseases. Highlights of the meeting included recent advances on the involvement of different epigenetic mechanisms in malignant and nonmalignant GI disorders and the epigenetic heterogeneity exhibited in these diseases. The translational value of epigenetic drugs, as well as the current and future use of epigenetic changes (i.e., DNA methylation patterns) as biomarkers for early detection tools or disease stratification were also important topics of discussion.

The availability of antigen tests for SARS-CoV-2 represents a major step for the mass surveillance of the incidence of infection, especially regarding COVID-19 asymptomatic and/or early-stage patients. Recently, we reported the development of a Bioelectric Recognition Assay-based biosensor able to detect the SARS-CoV-2 S1 spike protein expressed on the surface of the virus in just three minutes, with high sensitivity and selectivity. The working principle was established by measuring the change of the electric potential of membrane-engineered mammalian cells bearing the human chimeric spike S1 antibody after attachment of the respective viral protein. In the present study, we applied the novel biosensor to patient-derived nasopharyngeal samples in a clinical set-up, with absolutely no sample pretreatment. More importantly, membrane-engineered cells were pre-immobilized in a proprietary biomatrix, thus enabling their long-term preservation prior to use as well as significantly increasing their ease-of-handle as test consumables. The plug-and-apply novel biosensor was able to detect the virus in positive samples with a 92.8% success rate compared to RT-PCR. No false negative results were recorded. These findings demonstrate the potential applicability of the biosensor for the early, routine mass screening of SARS-CoV-2 on a scale not yet realized.

Background & Aims: Chronic inflammation promotes development and progression of colorectal cancer (CRC). We explored the distribution of the corticotropin-releasing-hormone (CRH) family of receptors and ligands in CRC and their contribution in tumor growth and oncogenic epithelial-to-mesenchymal transition (EMT). Methods: The mRNA expression of CRH-family members was analyzed in CRC (n = 56) and control (n = 46) samples, seven CRC cell lines, and normal NCM460 cells. Immunohistochemical detection of CRHR2 was performed in 20 CRC and five normal tissues. Cell proliferation, migration, and invasion were compared between urocortin-2 (Ucn2)-stimulated parental and CRHR2-overexpressing (CRHR2+) cells in the absence or presence of interleukin-6 (IL-6). CRHR2/Ucn2-targeted effects on tumor growth and EMT were validated in SW620-xenograft mouse models. Results: CRC tissues and cell lines showed decreased mRNA and protein CRHR2 expression compared with controls and NCM460 cells, respectively. The opposite trend was shown for Ucn2. CRHR2/Ucn2 signaling inhibited cell proliferation, migration, invasion, and colony formation in CRC-CRHR2+ cells. In vivo, SW620-CRHR2+ xenografts showed decreased growth, reduced expression of EMT-inducers, and elevated levels of EMT-suppressors. IL-1b, IL-6, and IL-6R mRNAs were diminished in CRC-CRHR2+ cells, while CRHR2/Ucn2 signaling inhibited IL-6-mediated Stat3 activation, invasion, migration, and expression of downstream targets acting as cell cycleâ and EMT-inducers. Expression of cell cycleâ and EMT-suppressors was augmented in IL-6/Ucn2-stimulated CRHR2+ cells. In patients, CRHR2 mRNA expression was inversely correlated with IL-6R and vimentin levels and metastasis occurrence, while positively associated with E-cadherin expression and overall survival. Conclusions: CRHR2 down-regulation in CRC supports tumor expansion and spread through maintaining persistent inflammation and constitutive Stat3 activation. CRHR2low CRC phenotypes are associated with higher risk for distant metastases and poor clinical outcomes. Keywords: Colorectal Cancer, Inflammation, Metastasis, Neuropeptides

Background & Aims: Substance P (SP), a neuropeptide member of the tachykinin family, plays a critical role in colitis. MicroRNAs (miRNAs) are small noncoding RNAs that negatively regulate gene expression. We examined whether SP modulates expression of microRNAs in human colonic epithelial cells. Methods: We performed microRNA profiling analysis of SP-stimulated human colonic epithelial NCM460 cells overexpressing neurokinin-1 receptor (NCM460-NK-1R). Targets of SP-regulated microRNAs were validated by real-time polymerase chain reaction (RT-PCR). Functions of miRNAs were tested in NCM460-NK-1R cells and the trinitrobenzene sulfonic acid (TNBS) and dextran sulfate sodium (DSS) models of colitis. Results: SP stimulated differential expression of 29 microRNAs, including miR-221-5p, the highest up-regulated miR (by 12.6-fold) upon SP stimulation. Bioinformatic and luciferase reporter analyses identified interleukin-6 receptor (IL-6R) mRNA as a direct target of miR-221-5p in NCM460 cells. Accordingly, SP exposure of NCM460-NK-1R cells increased IL-6R mRNA expression, and overexpression of miR-221-5p reduced IL-6R expression. Nuclear factor κB and c-Jun N-terminal kinase inhibition decreased SP-induced miR-221-5p expression. MiR-221-5p expression was increased in both TNBS- and DSS-induced colitis and in colonic biopsy samples from ulcerative colitis but not Crohnâs disease patients compared with controls. In mice, intracolonic administration of a miR-221-5p chemical inhibitor exacerbated TNBS- and DSS-induced colitis and increased colonic tumor necrosis factor-α, C-X-C motif chemokine 10 (Cxcl10), and collagen, type II, α 1 (Col2α1) mRNA expression. In situ hybridization in TNBS- and DSS-exposed colons revealed increased miR-221-5p expression primarily in colonocytes. Conclusions: Our results reveal a novel NK-1R-miR-221-5p-IL-6R network that protects from colitis. The use of miR-221-5p mimics may be a promising approach for colitis treatment. Keywords: Colitis, Inflammation, MicroRNA, Substance P

Pancreatic cancer is the fourth most frequent cause of cancer-related deaths and is characterized by early metastasis and pronounced resistance to chemotherapy and radiation therapy. Despite extensive esearch efforts, there is not any substantial progress regarding the identification of novel drugs against pancreatic cancer. Although the introduction of the chemotherapeutic agent gemcitabine improved clinical response, the prognosis of these patients remained extremely poor with a 5-year survival rate of 3-5%. Thus, the identification of the novel molecular pathways involved in pancreatic oncogenesis and the development of new and potent therapeutic options are highly desirable. Here, we describe how microRNAs control signaling pathways that are frequently deregulated during pancreatic oncogenesis. In addition, we provide evidence that microRNAs could be potentially used as novel pancreatic cancer therapeutics through reversal of chemotherapy and radiotherapy resistance or regulation of essential molecular pathways. Further studies should integrate the deregulated genes and microRNAs into molecular networks in order to identify the central regulators of pancreatic oncogenesis. Targeting these central regulators could lead to the development of novel targeted therapeutic approaches for pancreatic cancer patients.

We previously observed that treatment of mice with a dominant negative form of cJun (dn-cJun) increased the expression of genes involved in lipid metabolism and modulated the expression of nine microRNAs (miR). To investigate the potential effect of these miRs on the expression of the genes of lipid metabolism, we performed studies in cultured HepG2 cells. Transfection of HepG2 cells with sense or antisense miR-370 or miR-122 upregulated and downregulated, respectively, the transcription factor sterol-regulatory element binding protein 1c (SREBP-1c) and the enzymes diacylglycerol acyltransferase-2 (DGAT2), fatty acid synthase (FAS), and acyl-CoA carboxylase 1 (ACC1) that regulate fatty acid and triglyceride biosynthesis. The other seven miRs identified by the miR array screening did not affect the expression of lipogenic genes. miR-370 upregulated the expression of miR-122. Furthermore, the effect of miR-370 on the expression of the lipogenic genes was abolished by antisense miR-122. miR-370 targets the 3′ untranslated region (UTR) of Cpt1α, and it downregulated the expression of the carnitine palmitoyl transferase 1α (Cpt1α) gene as well as the rate of β oxidation. Our data suggest that miR-370 acting via miR-122 may have a causative role in the accumulation of hepatic triglycerides by modulating initially the expression of SREBP-1c, DGAT2, and Cpt1α and, subsequently, the expression of other genes that affect lipid metabolism.

Animal studies have shown that stress could induce epigenetic and transcriptomic alterations essential in determining the balance between adaptive or maladaptive responses to stress. We tested the hypothesis that chronic stress in rats deregulates coding and non-coding gene expression in the spinal cord, which may underline neuroinflammation and nociceptive changes previously observed in this model.Male Wistar rats were exposed to daily stress or handled, for 10 days. At day 11, lumbar spinal segments were collected and processed for mRNA/miRNA isolation followed by expression profiling using Agilent SurePrint Rat Exon and Rat miRNA Microarray platforms. Differentially expressed gene lists were generated using the dChip program. Microarrays were analyzed using the Ingenuity Pathways Analysis (IPA) tool from Ingenuity Systems. Multiple methods were used for the analysis of miRNA-mRNA functional modules. Quantitative real time RT-PCR for Interleukin 6 signal transducer (gp130), the Signal Transducer And Activator Of Transcription 3 (STAT3), glial fibrillary acidic protein and mir-17-5p were performed to confirm levels of expression.Gene network analysis revealed that stress deregulated different inflammatory (IL-6, JAK/STAT, TNF) and metabolic (PI3K/AKT) signaling pathways. MicroRNA array analysis revealed a signature of 39 deregulated microRNAs in stressed rats. MicroRNA-gene network analysis showed that microRNAs are regulators of two gene networks relevant to inflammatory processes. Specifically, our analysis of miRNA-mRNA functional modules identified miR-17-5p as an important regulator in our model. We verified miR-17-5p increased expression in stress using qPCR and in situ hybridization. In addition, we observed changes in the expression of gp130 and STAT3 (involved in intracellular signaling cascades in response to gp130 activation), both predicted targets for miR-17-5p. A modulatory role of spinal mir17-5p in the modulation of visceral sensitivity was confirmed in vivo.Using an integrative high throughput approach, our findings suggest a link between miR-17-5p increased expression and gp130/STAT3 activation providing new insight into the possible mechanisms mediating the effect of chronic stress on neuroinflammation in the spinal cord.

Recent studies have implied that osteoarthritis (OA) is a metabolic disease linked to deregulation of genes involved in lipid metabolism and cholesterol efflux. Sterol Regulatory Element Binding Proteins (SREBPs) are transcription factors regulating lipid metabolism with so far no association with OA. Our aim was to test the hypothesis that SREBP-2, a gene that plays a key role in cholesterol homeostasis, is crucially involved in OA pathogenesis and to identify possible mechanisms of action.We performed a genetic association analysis using a cohort of 1,410 Greek OA patients and healthy controls and found significant association between single nucleotide polymorphism (SNP) 1784G>C in SREBP-2 gene and OA development. Moreover, the above SNP was functionally active, as normal chondrocytes' transfection with SREBP-2-G/C plasmid resulted in interleukin-1β and metalloproteinase-13 (MMP-13) upregulation. We also evaluated SREBP-2, its target gene 3-hydroxy-3-methylglutaryl-coenzymeA reductase (HMGCR), phospho-phosphoinositide3-kinase (PI3K), phospho-Akt, integrin-alphaV (ITGAV) and transforming growth factor-β (TGF-β) mRNA and protein expression levels in osteoarthritic and normal chondrocytes and found that they were all significantly elevated in OA chondrocytes. To test whether TGF-β alone can induce SREBP-2, we treated normal chondrocytes with TGF-β and found significant upregulation of SREBP-2, HMGCR, phospho-PI3K and MMP-13. We also showed that TGF-β activated aggrecan (ACAN) in chondrocytes only through Smad3, which interacts with SREBP-2. Finally, we examined the effect of an integrin inhibitor, cyclo-RGDFV peptide, on osteoarthritic chondrocytes, and found that it resulted in significant upregulation of ACAN and downregulation of SREBP-2, HMGCR, phospho-PI3K and MMP-13 expression levels.We demonstrated, for the first time, the association of SREBP-2 with OA pathogenesis and provided evidence on the molecular mechanism involved. We suggest that TGF-β induces SREBP-2 pathway activation through ITGAV and PI3K playing a key role in OA and that integrin blockage may be a potential molecular target for OA treatment.

Gene profiling studies provide important information for key molecules relevant to a disease but are less informative of protein-protein interactions, post-translational modifications and regulation by targeted subcellular localization. Integration of genomic data and construction of functional gene networks may provide additional insights into complex diseases such as systemic lupus erythematosus (SLE).We analyzed gene expression microarray data of bone marrow mononuclear cells (BMMCs) from 20 SLE patients (11 with active disease) and 10 controls. Gene networks were constructed using the bioinformatic tool Ingenuity Gene Network Analysis. In SLE patients, comparative analysis of BMMCs genes revealed a network with 19 central nodes as major gene regulators including ERK, JNK, and p38 MAP kinases, insulin, Ca(2+) and STAT3. Comparison between active versus inactive SLE identified 30 central nodes associated with immune response, protein synthesis, and post-transcriptional modification. A high degree of identity between networks in active SLE and non-Hodgkin's lymphoma (NHL) patients was found, with overlapping central nodes including kinases (MAPK, ERK, JNK, PKC), transcription factors (NF-kappaB, STAT3), and insulin. In validation studies, western blot analysis in splenic B cells from 5-month-old NZB/NZW F1 lupus mice showed activation of STAT3, ITGB2, HSPB1, ERK, JNK, p38, and p32 kinases, and downregulation of FOXO3 and VDR compared to normal C57Bl/6 mice.Gene network analysis of lupus BMMCs identified central gene regulators implicated in disease pathogenesis which could represent targets of novel therapies in human SLE. The high similarity between active SLE and NHL networks provides a molecular basis for the reported association of the former with lymphoid malignancies.

BACKGROUND: Osteoarthritis is a multifactorial disease characterized by destruction of the articular cartilage due to genetic, mechanical and environmental components affecting more than 100 million individuals all over the world. Despite the high prevalence of the disease, the absence of large-scale molecular studies limits our ability to understand the molecular pathobiology of osteoathritis and identify targets for drug development. METHODOLOGY/PRINCIPAL FINDINGS: In this study we integrated genetic, bioinformatic and proteomic approaches in order to identify new genes and their collaborative networks involved in osteoarthritis pathogenesis. MicroRNA profiling of patient-derived osteoarthritic cartilage in comparison to normal cartilage, revealed a 16 microRNA osteoarthritis gene signature. Using reverse-phase protein arrays in the same tissues we detected 76 differentially expressed proteins between osteoarthritic and normal chondrocytes. Proteins such as SOX11, FGF23, KLF6, WWOX and GDF15 not implicated previously in the genesis of osteoarthritis were identified. Integration of microRNA and proteomic data with microRNA gene-target prediction algorithms, generated a potential "interactome" network consisting of 11 microRNAs and 58 proteins linked by 414 potential functional associations. Comparison of the molecular and clinical data, revealed specific microRNAs (miR-22, miR-103) and proteins (PPARA, BMP7, IL1B) to be highly correlated with Body Mass Index (BMI). Experimental validation revealed that miR-22 regulated PPARA and BMP7 expression and its inhibition blocked inflammatory and catabolic changes in osteoarthritic chondrocytes. CONCLUSIONS/SIGNIFICANCE: Our findings indicate that obesity and inflammation are related to osteoarthritis, a metabolic disease affected by microRNA deregulation. Gene network approaches provide new insights for elucidating the complexity of diseases such as osteoarthritis. The integration of microRNA, proteomic and clinical data provides a detailed picture of how a network state is correlated with disease and furthermore leads to the development of new treatments. This strategy will help to improve the understanding of the pathogenesis of multifactorial diseases such as osteoarthritis and provide possible novel therapeutic targets.