A mTurquoise-Based cAMP Sensor for Both FLIM and Ratiometric Read-Out Has Improved Dynamic Range

FRET-based sensors for cyclic Adenosine Mono Phosphate (cAMP) have revolutionized the way in which this important intracellular messenger is studied. The currently prevailing sensors consist of the cAMP-binding protein Epac1, sandwiched between suitable donor- and acceptor fluorescent proteins (FPs). Through a conformational change in Epac1, alterations in cellular cAMP levels lead to a change in FRET that is most commonly detected by either Fluorescence Lifetime Imaging (FLIM) or by Sensitized Emission (SE), e.g., by simple ratio-imaging. We recently reported a range of different Epac-based cAMP sensors with high dynamic range and signal-to-noise ratio. We showed that constructs with cyan FP as donor are optimal for readout by SE, whereas other constructs with green FP donors appeared much more suited for FLIM detection. In this study, we present a new cAMP sensor, termed TEpacVV, which employs mTurquoise as donor. Spectrally very similar to CFP, mTurquoise has about doubled quantum efficiency and unlike CFP, its fluorescence decay is strictly single-exponential. We show that TEpacVV appears optimal for detection both by FLIM and SE, that it has outstanding FRET span and signal-to-noise ratio, and improved photostability. Hence, TEpacVV should become the cAMP sensor of choice for new experiments, both for FLIM and ratiometric detection

Less adhesiolysis and hernia repair during completion proctocolectomy after laparoscopic emergency colectomy for ulcerative colitis

The aim of this study was to determine whether the need for adhesiolysis during completion proctectomy (CP) with ileopouch anal anastomosis (IPAA) is influenced by the surgical approach of the initial emergency colectomy for ulcerative colitis and the hospital setting. One hundred consecutive patients who underwent CP with IPAA in our center between January 1999 and April 2010 were included. Emergency colectomy had been performed laparoscopically in 30 of 52 patients at the Academic Medical Center Amsterdam and in 6 of 48 patients at referring hospitals. Case files of these patients were retrospectively reviewed. Significantly more extensive adhesiolysis was performed after open compared to laparoscopic colectomy (47 vs. 6%, P <0.001). In univariate analysis, emergency colectomy at a referring hospital was also predictive for adhesiolysis (P = 0.003), but the open approach for the initial colectomy was the only independent predictive factor for the need for adhesiolysis (P <0.001) in a multivariable ordinal logistic regression analysis. Operating time of CP was significantly longer when limited [18 (95% CI = 0-36) min] or extensive [55 (35-75) min] adhesiolysis had to be performed. The interval to CP was longer after open colectomy and after colectomy performed at a referring hospital. Significantly more incisional hernia corrections during CP were performed after open emergency colectomy (14 vs. 0%, P = 0.024). Overall morbidity and postoperative hospital stay of CP were not related to the surgical approach or the hospital setting of the emergency colectomy. Laparoscopic as opposed to open emergency colectomy is associated with less adhesiolysis, fewer incisional hernias, and a shorter interval to completion proctectom

Collagen proportionate area correlates with histological stage and predicts clinical events in primary sclerosing cholangitis

Background & Aims: Primary sclerosing cholangitis (PSC) is a chronic cholestatic liver disease in need of accurate biomarkers for stratification and as surrogates for clinical endpoints in trials. Quantitative liver fibrosis assessment by collagen proportionate area (CPA) measurement has been demonstrated to correlate with clinical outcomes in chronic hepatitis C, alcohol-related and non-alcoholic fatty liver disease. We aimed to investigate the ability of CPA to quantify liver fibrosis and predict clinical events in PSC. Methods: Biopsies from 101 PSC patients from two European centres were retrospectively assessed by two expert pathologists in tandem, using grading (Ishak and Nakanuma) and staging (Ishak, Nakanuma, Ludwig) systems recently validated to predict clinical events in PSC. CPA was determined by image analysis of picro-Sirius red-stained sections following a standard protocol. We assessed the correlations between CPA, staging and grading and their associations with three outcomes: (1) time to PSC-related death, liver transplant or primary liver cancer; (2) liver transplant-free survival; (3) occurrence of cirrhosis-related clinical manifestations. Results: CPA correlated strongly with histological stage determined by each scoring system (P < .001) and was significantly associated with the three endpoints. Median time to endpoint-1, endpoint-2 and endpoint-3 was shorter in patients with higher CPA, on Kaplan-Meier analyses (P = .011, P = .034 and P = .001, respectively). Conclusion: Quantitative fibrosis assessment by CPA has utility in PSC. It correlates with established histological staging systems and predicts clinical events. CPA may be a useful tool for staging fibrosis and for risk stratification in PSC and should be evaluated further within prospective clinical trials

Anaerobic Feces Processing for Fecal Microbiota Transplantation Improves Viability of Obligate Anaerobes

Fecal microbiota transplantation (FMT) is under investigation for several indications, including ulcerative colitis (UC). The clinical success of FMT depends partly on the engraftment of viable bacteria. Because the vast majority of human gut microbiota consists of anaerobes, the currently used aerobic processing protocols of donor stool may diminish the bacterial viability of transplanted material. This study assessed the effect of four processing techniques for donor stool (i.e., anaerobic and aerobic, both direct processing and after temporary cool storage) on bacterial viability. By combining anaerobic culturing on customized media for anaerobes with 16S rRNA sequencing, we could successfully culture and identify the majority of the bacteria present in raw fecal suspensions. We show that direct anaerobic processing of donor stool is superior to aerobic processing conditions for preserving the bacterial viability of obligate anaerobes and butyrate-producing bacteria related to the clinical response to FMT in ulcerative colitis patients, including Faecalibacterium, Eubacterium hallii, and Blautia. The effect of oxygen exposure during stool processing decreased when the samples were stored long-term. Our results confirm the importance of sample conditioning to preserve the bacterial viability of oxygen-sensitive gut bacteria. Anaerobic processing of donor stool may lead to increased clinical success of FMT, which should further be investigated in clinical trials.</p

Comparison of proactive and conventional treatment of anastomotic leakage in rectal cancer surgery:a multicentre retrospective cohort series

Purpose: Comparative studies on efficacy of treatment strategies for anastomotic leakage (AL) after low anterior resection (LAR) are almost non-existent. This study aimed to compare different proactive and conservative treatment approaches for AL after LAR. Methods: This retrospective cohort study included all patients with AL after LAR in three university hospitals. Different treatment approaches were compared, including a pairwise comparison of conventional treatment and endoscopic vacuum-assisted surgical closure (EVASC). Primary outcomes were healed and functional anastomosis rates at end of follow-up. Results: Overall, 103 patients were included, of which 59 underwent conventional treatment and 23 EVASC. Median number of reinterventions was 1 after conventional treatment, compared to 7 after EVASC (p &lt; 0.01). Median follow-up was 39 and 25 months, respectively. Healed anastomosis rate was 61% after conventional treatment, compared to 78% after EVASC (p = 0.139). Functional anastomosis rate was higher after EVASC, compared to conventional treatment (78% vs. 54%, p = 0.045). Early initiation of EVASC in the first week after primary surgery resulted in better functional anastomosis rate compared to later initiation (100% vs. 55%, p = 0.008). Conclusion: Proactive treatment of AL consisting of EVASC resulted in improved healed and functional anastomosis rates for AL after LAR for rectal cancer, compared to conventional treatment. If EVASC was initiated within the first week after index surgery, a 100% functional anastomosis rate was achievable.</p

Comparison of proactive and conventional treatment of anastomotic leakage in rectal cancer surgery:a multicentre retrospective cohort series

Purpose: Comparative studies on efficacy of treatment strategies for anastomotic leakage (AL) after low anterior resection (LAR) are almost non-existent. This study aimed to compare different proactive and conservative treatment approaches for AL after LAR. Methods: This retrospective cohort study included all patients with AL after LAR in three university hospitals. Different treatment approaches were compared, including a pairwise comparison of conventional treatment and endoscopic vacuum-assisted surgical closure (EVASC). Primary outcomes were healed and functional anastomosis rates at end of follow-up. Results: Overall, 103 patients were included, of which 59 underwent conventional treatment and 23 EVASC. Median number of reinterventions was 1 after conventional treatment, compared to 7 after EVASC (p &lt; 0.01). Median follow-up was 39 and 25 months, respectively. Healed anastomosis rate was 61% after conventional treatment, compared to 78% after EVASC (p = 0.139). Functional anastomosis rate was higher after EVASC, compared to conventional treatment (78% vs. 54%, p = 0.045). Early initiation of EVASC in the first week after primary surgery resulted in better functional anastomosis rate compared to later initiation (100% vs. 55%, p = 0.008). Conclusion: Proactive treatment of AL consisting of EVASC resulted in improved healed and functional anastomosis rates for AL after LAR for rectal cancer, compared to conventional treatment. If EVASC was initiated within the first week after index surgery, a 100% functional anastomosis rate was achievable.</p