Analysis of microsatellite instability in colorectal carcinoma by microfluidic-based chip electrophoresis

Microsatellite analysis is an important tool in clinical research and molecular diagnostics because microsatellite instability (MSI) occurs frequently in various types of cancer. Approximately 10–15% of colorectal, gastric and endometrial carcinomas are associated with MSI, and this has an impact on clinical prognosis. The microsatellite loci Bat25, Bat26, D2S123, D5S346 and D17S250, recommended by the Bethesda guidelines, were analysed by microfluidic-based on-chip electrophoresis in 40 cases of colon carcinoma with known MSI status. In all cases, microfluidic separation of the PCR amplicons resulted in highly resolved, distinct patterns of each of the five microsatellite loci. Detection of MSI could be demonstrated by microsatellite-loci-associated, well-defined deviations in the electropherogram profiles of tumour and non-tumour material, and confirmed the classification of MSI cases performed by conventional technology. In conclusion, microfluidic chip technology is a simple and reliable approach for MSI detection that allows label-free and very fast analysis of microsatellite amplicons

Site-specific perturbations of alpha-synuclein fibril structure by the Parkinson's disease associated mutations A53T and E46K.

PMCID: PMC3591419This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.Parkinson's disease (PD) is pathologically characterized by the presence of Lewy bodies (LBs) in dopaminergic neurons of the substantia nigra. These intracellular inclusions are largely composed of misfolded α-synuclein (AS), a neuronal protein that is abundant in the vertebrate brain. Point mutations in AS are associated with rare, early-onset forms of PD, although aggregation of the wild-type (WT) protein is observed in the more common sporadic forms of the disease. Here, we employed multidimensional solid-state NMR experiments to assess A53T and E46K mutant fibrils, in comparison to our recent description of WT AS fibrils. We made de novo chemical shift assignments for the mutants, and used these chemical shifts to empirically determine secondary structures. We observe significant perturbations in secondary structure throughout the fibril core for the E46K fibril, while the A53T fibril exhibits more localized perturbations near the mutation site. Overall, these results demonstrate that the secondary structure of A53T has some small differences from the WT and the secondary structure of E46K has significant differences, which may alter the overall structural arrangement of the fibrils

High-Resolution 3D Structure Determination of Kaliotoxin by Solid-State NMR Spectroscopy

High-resolution solid-state NMR spectroscopy can provide structural information of proteins that cannot be studied by X-ray crystallography or solution NMR spectroscopy. Here we demonstrate that it is possible to determine a protein structure by solid-state NMR to a resolution comparable to that by solution NMR. Using an iterative assignment and structure calculation protocol, a large number of distance restraints was extracted from 1H/1H mixing experiments recorded on a single uniformly labeled sample under magic angle spinning conditions. The calculated structure has a coordinate precision of 0.6 Å and 1.3 Å for the backbone and side chain heavy atoms, respectively, and deviates from the structure observed in solution. The approach is expected to be applicable to larger systems enabling the determination of high-resolution structures of amyloid or membrane proteins

Meta-analysis of extracorporeal membrane oxygenation in combination with intra-aortic balloon pump vs. extracorporeal membrane oxygenation only in patients with cardiogenic shock due to acute myocardial infarction

BackgroundIncidence and mortality of cardiogenic shock (CS) in patients with acute myocardial infarction (AMI) remain high despite substantial therapy improvements in acute percutaneous coronary intervention over the last decades. Unloading the left ventricle in patients with Veno-arterial extracorporeal membrane oxygenation (VA-ECMO) can be performed by using an intra-aortic balloon pumps’ (IABP) afterload reduction, which might be especially beneficial in AMI patients with CS.ObjectiveThe objective of this meta-analysis was to assess the effect of VA-ECMO + IABP vs. VA-ECMO treatment on the mortality of patients with CS due to AMI.MethodsA systematic literature search was performed using EMBASE, COCHRANE, and MEDLINE databases. Studies comparing the effect of VA-ECMO + IABP vs. VA-ECMO on mortality of patients with AMI were included. Meta-analyses were performed to analyze the effect of the chosen treatment on 30-day/in-hospital mortality.ResultsTwelve studies were identified by the literature search, including a total of 5,063 patients, 81.5% were male and the mean age was 65.9 years. One thousand one hundred and thirty-six patients received treatment with VA-ECMO in combination with IABP and 2,964 patients received VA-ECMO treatment only. The performed meta-analysis showed decreased mortality at 30-days/in-hospital after VA-ECMO + IABP compared to VA-ECMO only for patients with cardiogenic shock after AMI (OR 0.36, 95% CI 0.30–0.44, P≤0.001). Combination of VA-ECMO + IABP was associated with higher rates of weaning success (OR 0.29, 95% CI 0.16–0.53, P < 0.001) without an increase of vascular access complications (OR 0.85, 95% CI 0.35–2.08, P = 0.72).ConclusionIn this meta-analysis, combination therapy of VA-ECMO + IABP was superior to VA-ECMO only therapy in patients with CS due to AMI. In the absence of randomized data, these results are hypothesis generating only

Electrocardiographic changes during sustained normobaric hypoxia in patients after myocardial infarction

The safety of prolonged high-altitude stays and exercise for physically fit post-myocardial infarction (MI) patients is unclear. Myocardial tissue hypoxia and pulmonary hypertension can affect cardiac function and electrophysiology, possibly contributing to arrhythmias. We included four non-professional male athletes, clinically stable after left ventricular MI (three with ST-segment elevation MI and one with non-ST-segment elevation MI) treated with drug-eluting stents for single-vessel coronary artery disease. Oxygen levels were reduced to a minimum of 11.8%, then restored to 20.9%. We conducted electrocardiography (ECG), ergometry, and echocardiography assessments in normoxic and hypoxic conditions. With an average age of 57.8 ± 3.3 years and MI history 37 to 104 months prior, participants experienced a significant increase in QTc intervals during hypoxia using Bazett's (from 402 ± 13 to 417 ± 25 ms), Fridericia's (from 409 ± 12 to 419 ± 19 ms), and Holzmann's formulas (from 103 ± 4 to 107 ± 6%) compared to normoxia. This effect partially reversed during recovery. Echocardiographic signs of pulmonary hypertension during normobaric hypoxia correlated significantly with altered QTc intervals (p < 0.001). Despite good health and complete revascularization following MI, susceptibility to hypoxia-induced QTc prolongation and ventricular ectopic beats persists, especially during physical activity. MI survivors planning high-altitude activities should consult cardiovascular specialists with high-altitude medicine expertise

Dynamics of cancer cell subpopulations in primary and metastatic colorectal tumors

Intratumor heterogeneity—heterogeneity of cancer cells within a single tumor—is considered one of the most problematic factors of treatment. Genetic heterogeneity, such as in somatic mutations and chromosome aberrations, is a common characteristic of human solid tumors and is probably the basis of biological heterogeneity. Using mutations in APC, TP53 and KRAS as markers to identify distinct colorectal cancer subpopulations, we analyzed a total of 42 primary colorectal cancer tissues and six paired liver metastases with multipoint microsampling, which enabled analysis of mutation patterns and allelic imbalances with a resolution of 0.01 mm2 (about 200 cells). There was usually more than one subpopulation in each primary tumor. Only two of 15 (13.3%) cases with three gene mutations and eight of 27 (29.6%) cases with two gene mutations had a single subpopulation. Cells with mutations in all of the examined genes usually constituted the major population. Multipoint microsampling of six primary and metastatic tumor pairs revealed that the majority of discrepancies in mutation patterns found with the bulk tissue analysis were due to loss of subpopulations in the metastatic tissues. In addition, multipoint microsampling uncovered substantial changes in subpopulations that were not detected with bulk tissue analysis. Specifically, the proportion of KRAS mutation-negative subpopulations increased in the metastatic tumors of four cases. Because KRAS mutation status is linked to cetuximab/panitumumab efficacy, subpopulation dynamics could lead to differences in response to cetuximab/panitumumab in primary versus metastatic tumors