Direct isolation of small extracellular vesicles from human blood using viscoelastic microfluidics.

Small extracellular vesicles (sEVs; <200 nm) that contain lipids, nucleic acids, and proteins are considered promising biomarkers for a wide variety of diseases. Conventional methods for sEV isolation from blood are incompatible with routine clinical workflows, significantly hampering the utilization of blood-derived sEVs in clinical settings. Here, we present a simple, viscoelastic-based microfluidic platform for label-free isolation of sEVs from human blood. The separation performance of the device is assessed by isolating fluorescent sEVs from whole blood, demonstrating purities and recovery rates of over 97 and 87%, respectively. Significantly, our viscoelastic-based microfluidic method also provides for a remarkable increase in sEV yield compared to gold-standard ultracentrifugation, with proteomic profiles of blood-derived sEVs purified by both methods showing similar protein compositions. To demonstrate the clinical utility of the approach, we isolate sEVs from blood samples of 20 patients with cancer and 20 healthy donors, demonstrating that elevated sEV concentrations can be observed in blood derived from patients with cancer.

Tissue and Circulating MicroRNA-31, MicroRNA-200b, and MicroRNA-200c Reflects Disease Activity in Crohn's Disease Patients: Results from the BIOMIR Study.

BACKGROUND AND AIMS: MicroRNAs (miR) have altered expression in multiple autoimmune disorders including inflammatory bowel disease. The aim of the study was to assess the tissue and circulating miR-31, miR-200b, and miR-200c expression levels as potential biomarkers for intestinal disease activity in patients with Crohn's disease (CD). METHODS: The study included 45 patients with histopathological confirmed CD and active disease (defined as fecal calprotectin >50 µg/g and Simple Endoscopic Score (SES) of CD >3), and 21 subjects as controls for the validation cohort. Demographic and clinical data, biomarkers (fecal calprotectin), endoscopy data, the expression levels of miR-31, miR-200b, and miR-200c in tissue and serum were assessed (by RT-PCR). Receiver operating characteristic analysis was performed to assess the miR-31, miR-200b, and miR-200c expression levels as potential biomarkers for active CD. RESULTS: Mean fecal calprotectin was 1540±890 µg/g. Mean SES-CD was 8.9±4.2. Tissue and circulating miR- 31 were significantly correlated with fecal calprotectin (r=0.81, r=0.83, p<0.01) and with SES-CD (r=0.82, r=0.79, p<0.01). The expression level of miR-31 was significantly upregulated in CD tissue cases compared to the control tissue samples (6.24±1.57 vs. 3.70±1.44; p <0.01). Similarly, serum miR-31 expression levels in CD patients were significantly upregulated compared to the control serum samples (0.78±0.42 vs. -2.07±1.00; p<0.01). The expression levels of tissue miR-200b and miR-200c were significantly upregulated in CD tissue cases compared to the control tissue samples (-5.25±0.93 vs. -4.69±0.80, p=0.03 for miR-200b, and -0.86±0.96 vs. 0.39±0.66, p<0.01 for miR-200c). Similarly, serum miR-200b and miR-200c expression levels in CD patients were significantly upregulated compared to the control serum samples (p < 0.05). Receiver operating characteristic analysis revealed that the expression levels of the selected miRNAs could help to discriminate active CD patients from healthy controls with very good specificity and sensitivity. CONCLUSIONS: Tissue and circulating miR-31, miR-200b, and miR-200c reflect disease activity in CD patients and can be used as biomarkers for active disease.

The Role of Artificial Intelligence in Monitoring Inflammatory Bowel Disease-The Future Is Now.

Crohn's disease and ulcerative colitis remain debilitating disorders, characterized by progressive bowel damage and possible lethal complications. The growing number of applications for artificial intelligence in gastrointestinal endoscopy has already shown great potential, especially in the field of neoplastic and pre-neoplastic lesion detection and characterization, and is currently under evaluation in the field of inflammatory bowel disease management. The application of artificial intelligence in inflammatory bowel diseases can range from genomic dataset analysis and risk prediction model construction to the disease grading severity and assessment of the response to treatment using machine learning. We aimed to assess the current and future role of artificial intelligence in assessing the key outcomes in inflammatory bowel disease patients: endoscopic activity, mucosal healing, response to treatment, and neoplasia surveillance.

A plea for a unified approach to sedation in gastrointestinal endoscopy in Romania: results from a prospective multicentric trial.

Background. Adequate sedation is a prerequisite for quality endoscopic examination of the digestive tract. We aimed to evaluate the current practices and safety profile of sedation for gastrointestinal endoscopy in Romania and its impact on the technical success of the procedure and procedure-related adverse events. Methods. We conducted a prospective, multicentric, observational study including all patients undergoing digestive endoscopic procedures under various degrees of sedation. We collected data regarding the endoscopic procedure, type and degree of sedation, drug regimens, personnel in charge of sedation, and relevant patient related information. The main study outcome was the rate of sedation-related adverse events; secondary study outcomes included procedure-related adverse events and the impact of sedation on procedure success. Results. 1,043 consecutive endoscopic procedures from eight Romanian endoscopy units were included in our study. Sedation regimens were highly variable between participating centers, with 566 (54%) of procedures being performed under sedation provided by an anaesthesiologist. Sedation-related adverse events occurred in 40 cases (3.8%), most of them were mild respiratory and cardiovascular events and all reversed spontaneously. On multivariate analysis, male gender, procedure type (endoscopic ultrasound and endoscopic retrograde cholangiopancreatography) and deep sedation were risk factors for complications. The endoscopy unit, ASA status, age and type of sedative did not influence the complication rate. Conclusion. In conclusion, sedation for endoscopic procedures is generally safe, despite a high variability in sedation practices between centers in Romania. Establishing a national guideline on sedation for gastrointestinal endoscopy will ensure consistent and safe practice for these procedures.

Unmet needs in the diagnosis and treatment of Romanian patients with bilio-pancreatic tumors: results of a prospective observational multicentric study.

Background. Biliopancreatic tumors (BPT) are among the most aggressive solid malignancies, and their incidence is rising. Good patient outcome relies heavily on a multidisciplinary approach to therapy, including timely access to endoscopy, surgery and chemo/radiotherapy. We aimed to evaluate current practices as reflected in the management and outcome of patients diagnosed with BPT in the setting of a low-resource medical system in order to identify areas suitable for improvement. Material and methods. We conducted a prospective observational study of patients with pancreatic cancers and extrahepatic cholangiocarcinomas evaluated in 4 referral centers in Romania. We collected data on the pathology of the tumors, staging at diagnosis, ECOG status, surgical interventions, chemo/radiotherapy and endoscopic drainage where applicable. A telephonic follow-up visit at 3 months after the enrollment visit collected additional data regarding evolution, subsequent treatment, performance status and disease-related events and outcomes. Results and conclusions. One hundred seventy-two patients were included in the study during a one-year period at the four participating centers. 72.1% were diagnosed with pancreatic cancer while 27.9% had extrahepatic cholangiocarcinoma. We identified several unmet needs in the current practices of treatment for these malignancies: a lack of pathological confirmation in 25.6% of the cases, a very low percentage of resectable lesions (only 18% of the patients operated with curative intent), and suboptimal choice of drainage in patients who required palliative drainage at their first endoscopic intervention. Significant effort is required to ensure standard-of-care treatment for patient with BPT in low-resource medical systems, including comprehensive auditing and protocol surveillance.

SRED-ARCE Recommendations for Minimally Invasive Interventions During the COVID-19 Pandemic in Romania.

The Romanian Society of Digestive Endoscopy (SRED) and the Romanian Association of Endoscopic Surgery (ARCE) have decided to establish a joint working group to elaborate specific recommendations for organizing the diagnostic and the minimally invasive interventional procedures, in the context of the COVID-19 pandemic. The recommendations are based on the guidelines of the international societies of endoscopy and gastroenterology (ESGE / BSG / ASGE / ACG / AGA), respectively endoscopic surgery (EAES SAGES) (4-8), on the experience of countries severely affected by the pandemic (Italy, France, Spain, USA, Germany, etc.) and they will be applied within the limits of measures imposed at local and governmental level by the competent authorities. On the other hand, these recommendations should have a dynamic evolution, depending on the upward or downward trend of the COVID-19 pandemic at regional and local level, but also according to the findings of professional and academic societies, requiring regular reviews based on the publica tion of further recommendations or international clinical trials. The objectives of the SRED and ARCE recommendations target the endoscopic and laparoscopic surgery activities, to support their non discriminatory used for diagnostic or therapeutic purposes, pursuing the demonstrated benefits of these procedures, in safe conditions for patients and medical staff.

Endoscopy in inflammatory bowel disease: from guidelines to real life.

Endoscopy has a central role in the management of inflammatory bowel disease (IBD), providing crucial data for diagnostic and therapeutic decisions, treating disease-related complications, and assisting in the early detection of dysplasia and prevention of colorectal cancer in the setting of IBD. Treatment targets have significantly shifted in IBD, focusing on achieving mucosal healing, a more meaningful endpoint than clinical remission. With the emergence of novel therapies, we aim to alter the course of the disease and prevent irreversible damage to the bowel. To that end, obtaining reliable and reproducible assessments of endoscopic disease activity has become an issue of great importance. Although several guidelines include recommendations regarding endoscopic surveillance in patients with long-standing IBD, there is an open debate regarding the best examination method and the appropriate follow-up intervals. Another important issue is whether surveillance guidelines are actually implemented in real-life practice and what is the preferred surveillance method among endoscopists. Significant changes have occurred in the endoscopic world with the development of new diagnostic and therapeutic modalities and their incorporation in everyday practice. We aimed to assess the real-life application of guideline recommendations regarding endoscopy in IBD patients and to review newly emerged data which might impact these recommendations in the near future.

Environmental Fate of RNA Interference Pesticides: Adsorption and Degradation of Double-Stranded RNA Molecules in Agricultural Soils.

Double-stranded RNA (dsRNA) pesticides are a new generation of crop protectants that interfere with protein expression in targeted pest insects by a cellular mechanism called RNA interference (RNAi). The ecological risk assessment of these emerging pesticides necessitates an understanding of the fate of dsRNA molecules in receiving environments, among which agricultural soils are most important. We herein present an experimental approach using phosphorus-32 (32P)-radiolabeled dsRNA that allows studying key fate processes of dsRNA in soils with unprecedented sensitivity. This approach resolves previous analytical challenges in quantifying unlabeled dsRNA and its degradation products in soils. We demonstrate that 32P-dsRNA and its degradation products are quantifiable at concentrations as low as a few nanograms of dsRNA per gram of soil by both Cerenkov counting (to quantify total 32P-activity) and by polyacrylamide gel electrophoresis followed by phosphorimaging (to detect intact 32P-dsRNA and its 32P-containing degradation products). We show that dsRNA molecules added to soil suspensions undergo adsorption to soil particle surfaces, degradation in solution, and potential uptake by soil microorganisms. The results of this work on dsRNA adsorption and degradation advance a process-based understanding of the fate of dsRNA in soils and will inform ecological risk assessments of emerging dsRNA pesticides.

Regulation of miR-200c/141 expression by intergenic DNA-looping and transcriptional read-through.

The miR-200 family members have been implicated in stress responses and ovarian tumorigenesis. Here, we find that miR-200c/141 transcription is intimately linked to the transcription of the proximal upstream gene PTPN6 (SHP1) in all physiological conditions tested. PTPN6 and miR-200c/141 are transcriptionally co-regulated by two complementary mechanisms. First, a bypass of the regular PTPN6 polyadenylation signal allows the transcription of the downstream miR-200c/141. Second, the promoters of the PTPN6 and miR-200c/141 transcription units physically interact through a 3-dimensional DNA loop and exhibit similar epigenetic regulation. Our findings highlight that transcription of intergenic miRNAs is a novel outcome of transcriptional read-through and reveal a yet unexplored type of DNA loop associating two closely located promoters. These mechanisms have significant relevance in ovarian cancers and stress response, pathophysiological conditions in which miR-200c/141 exert key functions.

Romanian guidelines on the diagnosis and treatment of exocrine pancreatic insufficiency.

In assessing exocrine pancreatic insufficiency (EPI), its diverse etiologies and the heterogeneous population affected should be considered. Diagnosing this condition remains a challenge in clinical practice especially for mild-to-moderate EPI, with the support of the time-consuming breath test or the coefficient of fat absorption. The fecal elastase-1 test, less precise for the diagnosis, cannot be useful for assessing treatment efficacy. Pancreatic enzyme replacement therapy (PERT) is the mainstay of treatment, whereby enteric-coated mini-microspheres are taken with every meal, in progressive doses based on an individual's weight and clinical symptoms. The main indication for PERT is chronic pancreatitis, in patients who have clinically relevant steatorrhea, abnormal pancreatic function test or abnormal function tests associated with symptoms of malabsorption such as weight loss or meteorism. While enzyme replacement therapy is not recommended in the initial stages of acute pancreatitis, pancreatic exocrine function should be monitored for at least 6-18 months. In the case of unresectable pancreatic cancer, replacement enzyme therapy helps to maintain weight and improve overall quality of life. It is also indicated in patients with celiac disease, who have chronic diarrhea (in spite of gluten-free diet), and in patients with cystic fibrosis with proven EPI.

Rapid fecal calprotectin level assessment and the SIBDQ score can accurately detect active mucosal inflammation in IBD patients in clinical remission: a prospective study.

BACKGROUND AND AIMS: Mucosal healing is an important predictor of disease-related outcome in patients with inflammatory bowel disease (IBD) patients, including those in clinical remission. However, colonoscopy is an invasive procedure and many patients decline repeated endoscopic examinations. We aimed to assess whether noninvasive biomarkers could accurately detect endoscopic mucosal inflammatory activity in IBD patients in clinical remission. METHODS: We conducted a prospective observational cohort study on IBD patients in clinical remission at Colentina Hospital, Bucharest. Clinical activity was assessed using the Mayo score and Crohn's Disease Activity Index (CDAI), quality of life was assessed using the Short Inflammatory Bowel Disease Questionnaire (SIBDQ). Serum C-reactive protein (CRP) and fecal calprotectin (FC) levels were determined. All patients underwent ileo-colonoscopy to assess mucosal inflammatory activity. RESULTS: 48 patients were included in this study, with 67% showing endoscopic disease activity. SIBD questionnaire and FC performed well as noninvasive markers of intestinal inflammation (AUROC 0.78 and 0.77, respectively), while CRP could not accurately predict endoscopic disease activity. Fecal calprotectin levels > 30 µg/g showed a 93% sensitivity and a 50% specificity for detecting inflammatory changes of the mucosa while a combined test using FC > 30µg/g and a SIBDQ score < 6 achieved 81.2% sensitivity and 75% specificity, respectively, in detecting active endoscopic disease. CONCLUSION: Fecal calprotectin and SIBDQ have good diagnostic accuracy in detecting mucosal inflammatory changes in IBD patients in clinical remission. Combining simple, noninvasive tests such as the SIBDQ and FC levels appears to be a practical method for monitoring disease activity in these patients, possibly reducing the need for repeat endoscopic examinations.

Serum endocan levels are increased in patients with inflammatory bowel disease.

BACKGROUND: Endocan is a marker of angiogenesis previously studied in various types of cancer and inflammatory conditions. Its expression is influenced by vascular endothelial growth factor A (VEGF A) and tumor necrosis factor alpha (TNF alpha), cytokines involved in pathogenetic pathways in inflammatory bowel disease (IBD). The aim of this study was to determine whether serum endocan levels were increased in IBD patients. METHODS: We conducted an exploratory pilot study. Serum endocan levels were determined in a group of 33 consecutive IBD patients from an observational cohort study ongoing at Colentina Hospital and compared to levels determined in two control groups: healthy controls and stage IV cancer patients. RESULTS: Endocan levels were significantly higher in the IBD group as compared to both healthy controls (p < 0.001) and cancer patients (p < 0.01). There was no correlation found between endocan levels and disease activity as assessed by clinical or endoscopical activity scores. CONCLUSIONS: There is a potential role for endocan in future biomarker studies in IBD patients.

Deletion of the activated protein-1 transcription factor JunD induces oxidative stress and accelerates age-related endothelial dysfunction.

BACKGROUND: Reactive oxygen species are major determinants of vascular aging. JunD, a member of the activated protein-1 family of transcription factors, is emerging as a major gatekeeper against oxidative stress. However, its contribution to reactive oxygen species homeostasis in the vasculature remains unknown. METHODS AND RESULTS: Endothelium-dependent vasorelaxation was impaired in young and old JunD(-/-) mice (6 and 22 months old) compared with age-matched wild-type mice. JunD(-/-) mice displayed an age-independent decline in endothelial nitric oxide release and endothelial nitric oxide synthase activity and increased mitochondrial superoxide formation and peroxynitrite levels. Furthermore, vascular expression and activity of the free radical scavengers manganese and extracellular superoxide dismutase and aldehyde dehydrogenase 2 were reduced, whereas the NADPH oxidase subunits p47phox, Nox2, and Nox4 were upregulated. These redox changes were associated with premature vascular aging, as shown by reduced telomerase activity, increased ß-galactosidase-positive cells, upregulation of the senescence markers p16(INK4a) and p53, and mitochondrial disruption. Interestingly, old wild-type mice showed a reduction in JunD expression and transcriptional activity resulting from promoter hypermethylation and binding with tumor suppressor menin, respectively. In contrast, JunD overexpression blunted age-induced endothelial dysfunction. In human endothelial cells, JunD knockdown exerted a similar impairment of the O2(-)/nitric oxide balance that was prevented by concomitant NADPH inhibition. In parallel, JunD expression was reduced in monocytes from old versus young healthy subjects and correlated with mRNA levels of scavenging and oxidant enzymes. CONCLUSIONS: JunD provides protection in aging-induced endothelial dysfunction and may represent a novel target to prevent reactive oxygen species-driven vascular aging.

First Pillcam Colon 2 capsule images of Whipple's disease: Case report and review of the literature.

Whipple's disease is a rare chronic systemic infection determined by the Gram-positive bacillus Tropheryma whipplei. The infection usually mainly involves the small bowel, but sometimes other organs are affected as well. Since the current standard clinical and biological tests are nonspecific, diagnosis is very difficult and relies on histopathology. Here we present the case of a 52-year-old man with chronic diarrhea and weight loss whose symptoms had been evolving for 2 years and whose diagnosis came unexpectedly after capsule examination. Diagnosis was confirmed by the histopathologic examination of endoscopic biopsy samples, and treatment with co-trimoxazole resulted in remission of symptoms. We present the first images of Whipple's disease obtained with the Pillcam Colon 2 video capsule system.

miR-141 and miR-200a act on ovarian tumorigenesis by controlling oxidative stress response.

Although there is evidence that redox regulation has an essential role in malignancies, its impact on tumor prognosis remains unclear. Here we show crosstalk between oxidative stress and the miR-200 family of microRNAs that affects tumorigenesis and chemosensitivity. miR-141 and miR-200a target p38α and modulate the oxidative stress response. Enhanced expression of these microRNAs mimics p38α deficiency and increases tumor growth in mouse models, but it also improves the response to chemotherapeutic agents. High-grade human ovarian adenocarcinomas that accumulate miR-200a have low concentrations of p38α and an associated oxidative stress signature. The miR200a-dependent stress signature correlates with improved survival of patients in response to treatment. Therefore, the role of miR-200a in stress could be a predictive marker for clinical outcome in ovarian cancer. In addition, although oxidative stress promotes tumor growth, it also sensitizes tumors to treatment, which could account for the limited success of antioxidants in clinical trials.

Interstitial cells of Cajal in the gut--a gastroenterologist's point of view.

Alterations of normal function of interstitial cells of Cajal (ICC) are reported in many intestinal disorders. Diagnosis of their involvement is rare (infrequent), but necessary to propose a specific treatment. This article reviews the place of ICC in the pathogenesis of achalasia, gastroesophageal reflux disease, infantile hypertrophic pyloric stenosis, chronic intestinal pseudo-obstruction and slow transit constipation. Moreover we discuss the role of the Cajal cells in the development of stromal tumors of the gastrointestinal tract.

Oxidative stress contributes to aging by enhancing pancreatic angiogenesis and insulin signaling.

JunD, a transcription factor of the AP-1 family, protects cells against oxidative stress. Here, we show that junD(-/-) mice exhibit features of premature aging and shortened life span. They also display persistent hypoglycemia due to enhanced insulin secretion. Consequently, the insulin/IGF-1 signaling pathways are constitutively stimulated, leading to inactivation of FoxO1, a positive regulator of longevity. Hyperinsulinemia most likely results from enhanced pancreatic islet vascularization owing to chronic oxidative stress. Indeed, accumulation of free radicals in beta cells enhances VEGF-A transcription, which in turn increases pancreatic angiogenesis and insulin secretion. Accordingly, long-term treatment with an antioxidant rescues the phenotype of junD(-/-) mice. Indeed, dietary antioxidant supplementation was protective against pancreatic angiogenesis, hyperinsulinemia, and subsequent activation of insulin signaling cascades in peripheral tissues. Taken together, these data establish a pivotal role for oxidative stress in systemic regulation of insulin and define a key role for the JunD protein in longevity.

An injected bacterial effector targets chromatin access for transcription factor NF-kappaB to alter transcription of host genes involved in immune responses.

Phosphorylation of histone H3 at Ser10 increases chromatin accessibility to transcription factor NF-kappaB on a subset of genes involved in immune responses. Here we report that a bacterial pathogen abrogated phosphorylation of histone H3 to 'shape' the transcriptional responses of infected host cells. We identify the Shigella flexneri protein effector OspF as a dually specific phosphatase that dephosphorylated mitogen-activated protein kinases in the nucleus, thus preventing histone H3 phosphorylation at Ser10 in a gene-specific way. That activity of OspF enabled shigella to block the activation of a subset of NF-kappaB-responsive genes, leading to compromised recruitment of polymorphonuclear leukocytes to infected tissues. S. flexneri has thus evolved the capacity to precisely modulate host cell epigenetic 'information' as a strategy for repressing innate immunity.