Rapid and Sensitive Lentivirus Vector-Based Conditional Gene Expression Assay to Monitor and Quantify Cell Fusion Activity

Cell-to-cell fusion is involved in multiple fundamental biological processes. Prominent examples include osteoclast and giant cell formation, fertilization and skeletal myogenesis which involve macrophage, sperm-egg and myoblast fusion, respectively. Indeed, the importance of cell fusion is underscored by the wide range of homeostatic as well as pathologic processes in which it plays a key role. Therefore, rapid and sensitive systems to trace and measure cell fusion events in various experimental systems are in demand. Here, we introduce a bipartite cell fusion monitoring system based on a genetic switch responsive to the site-specific recombinase FLP. To allow flexible deployment in both dividing as well as non-dividing cell populations, inducer and reporter modules were incorporated in lentivirus vector particles. Moreover, the recombinase-inducible transcription units were designed in such a way as to minimize basal activity and chromosomal position effects in the “off” and “on” states, respectively. The lentivirus vector-based conditional gene expression assay was validated in primary human mesenchymal stem cells and in a differentiation model based on muscle progenitor cells from a Duchenne muscular dystrophy patient using reporter genes compatible with live- and single-cell imaging and with whole population measurements. Using the skeletal muscle cell differentiation model, we showed that the new assay displays low background activity, a 2-log dynamic range, high sensitivity and is amenable to the investigation of cell fusion kinetics. The utility of the bipartite cell fusion monitoring system was underscored by a study on the impact of drug- and RNAi-mediated p38 MAPK inhibition on human myocyte differentiation. Finally, building on the capacity of lentivirus vectors to readily generate transgenic animals the present FLP-inducible system should be adaptable, alone or together with Cre/loxP-based assays, to cell lineage tracing and conditional gene manipulation studies in vivo

Discerning natural and anthropogenic organic matter inputs to salt marsh sediments of Ria Formosa lagoon (South Portugal)

Sedimentary organic matter (OM) origin and molecular composition provide useful information to understand carbon cycling in coastal wetlands. Core sediments from threors' Contributionse transects along Ria Formosa lagoon intertidal zone were analysed using analytical pyrolysis (Py-GC/MS) to determine composition, distribution and origin of sedimentary OM. The distribution of alkyl compounds (alkanes, alkanoic acids and alkan-2-ones), polycyclic aromatic hydrocarbons (PAHs), lignin-derived methoxyphenols, linear alkylbenzenes (LABs), steranes and hopanes indicated OM inputs to the intertidal environment from natural-autochthonous and allochthonous-as well as anthropogenic. Several n-alkane geochemical indices used to assess the distribution of main OM sources (terrestrial and marine) in the sediments indicate that algal and aquatic macrophyte derived OM inputs dominated over terrigenous plant sources. The lignin-derived methoxyphenol assemblage, dominated by vinylguaiacol and vinylsyringol derivatives in all sediments, points to large OM contribution from higher plants. The spatial distributions of PAHs (polyaromatic hydrocarbons) showed that most pollution sources were mixed sources including both pyrogenic and petrogenic. Low carbon preference indexes (CPI > 1) for n-alkanes, the presence of UCM (unresolved complex mixture) and the distribution of hopanes (C-29-C-36) and steranes (C-27-C-29) suggested localized petroleum-derived hydrocarbon inputs to the core sediments. Series of LABs were found in most sediment samples also pointing to domestic sewage anthropogenic contributions to the sediment OM.EU Erasmus Mundus Joint Doctorate fellowship (FUECA, University of Cadiz, Spain)EUEuropean Commission [FP7-ENV-2011, 282845, FP7-534 ENV-2012, 308392]MINECO project INTERCARBON [CGL2016-78937-R]info:eu-repo/semantics/publishedVersio

Effect of sitagliptin on cardiovascular outcomes in type 2 diabetes

BACKGROUND: Data are lacking on the long-term effect on cardiovascular events of adding sitagliptin, a dipeptidyl peptidase 4 inhibitor, to usual care in patients with type 2 diabetes and cardiovascular disease. METHODS: In this randomized, double-blind study, we assigned 14,671 patients to add either sitagliptin or placebo to their existing therapy. Open-label use of antihyperglycemic therapy was encouraged as required, aimed at reaching individually appropriate glycemic targets in all patients. To determine whether sitagliptin was noninferior to placebo, we used a relative risk of 1.3 as the marginal upper boundary. The primary cardiovascular outcome was a composite of cardiovascular death, nonfatal myocardial infarction, nonfatal stroke, or hospitalization for unstable angina. RESULTS: During a median follow-up of 3.0 years, there was a small difference in glycated hemoglobin levels (least-squares mean difference for sitagliptin vs. placebo, -0.29 percentage points; 95% confidence interval [CI], -0.32 to -0.27). Overall, the primary outcome occurred in 839 patients in the sitagliptin group (11.4%; 4.06 per 100 person-years) and 851 patients in the placebo group (11.6%; 4.17 per 100 person-years). Sitagliptin was noninferior to placebo for the primary composite cardiovascular outcome (hazard ratio, 0.98; 95% CI, 0.88 to 1.09; P<0.001). Rates of hospitalization for heart failure did not differ between the two groups (hazard ratio, 1.00; 95% CI, 0.83 to 1.20; P = 0.98). There were no significant between-group differences in rates of acute pancreatitis (P = 0.07) or pancreatic cancer (P = 0.32). CONCLUSIONS: Among patients with type 2 diabetes and established cardiovascular disease, adding sitagliptin to usual care did not appear to increase the risk of major adverse cardiovascular events, hospitalization for heart failure, or other adverse events

Optimization of SPECT/CT based lung dose calculation for Holmium-166 hepatic radioembolization

Background: Quantitative SPECT of the lungs after intra-arterial hepatic radioembolization using Holmium-166 (166Ho)microspheres is essential to assess therapy safety. A SPECT estimated lung absorbed dose resulting from radioembolization of more than 30 Gy is a contra-indication for therapy. Earlier we showed the superiority of Monte Carlo-based iterative reconstructions over conventional reconstructions due to its quantitative nature, required for dosimetry, at the cost of substantial computation times. In clinical routine, however, the limited available time between scout imaging and therapy constrains its application. To reduce computation times, we investigated the minimum number of iterations required to guarantee a clinical acceptable accuracy in lung dose estimation using patient and phantom data. Methods: 166Ho scout SPECT data (range: 222-283 MBq) were used from 9 patients. SPECT images were Monte Carlo-based OSEM reconstructed (iterations: 30, subsets: 8). Additionally, the 4D XCAT anthropomorphic phantom was used to mimic SPECT studies with an injected scout activity of 250 MBq and with varying lung doses ranging from 0 to 15.6 mGy/MBq. These studies were reconstructed in the same way as the patient data. Results: In all patients the lung absorbed dose upon OSEM convergence ranged from 0 to 0.025 mGy/MBq, and ranged from 0.002 to 0.078 mGy/MBq after five iterations, still well below the allowed 30 Gy in case treatment was proceeded. In the phantom data, the estimated lung dose ranged from 0.004 to 15 mGy/MBq upon convergence and from 0.03 to 13.9 mGy/MBq after five iterations, simulating situations well below and above an estimated treatment lung dose of 30 Gy. Importantly, the lung absorbed dose upon OSEM convergence was underestimated by 15% as compared to the actual simulated lung dose, and the dose after five OSEM iterations was underestimated by 9% as compared to the dose upon convergence. Both underestimations were irrespective of the magnitude of the lung dose and thus can be easily corrected for. Conclusions: The number of OSEM iterations necessary for a quantitative estimate of the lung dose can be reduced from 30 to 5. The resulting six fold reduction in calculation time enables data processing of the scout images before therapy administration

In vivo cardiac P-31 MRS in a mouse model of heart failure

Objective: To investigate myocardial energy status in a mouse model of heart failure using in vivo31P magnetic resonance spectroscopy (MRS). Methods: Male C57BL/6 mice underwent thoracic aortic constriction (TAC) surgery, inducing pressure-overload cardiomyopathy, and were measured seven weeks after surgery (n = 5). Healthy wild-type mice served as controls (n = 4). Cardiac cine 1H MR images were made for reference purposes and to quantify left ventricular (LV) function. Cardiac 31P MR spectra were measured from a ~6 mm cubic voxel enclosing the end-diastolic LV myocardium using ECG triggered, respiratory gated 3D Image-Selected In vivo Spectroscopy (ISIS). Results: LV end-diastolic volume and LV mass normalized to body weight were higher in TAC mice compared to controls (91.7 ± 19.0 versus 61.8 ± 6.0 µL and 4.4 ± 0.6 versus 3.1 ± 0.2 mg/g, P &lt;0.01), whereas LV ejection fraction was reduced in TAC mice (45.4 ± 20.0 versus 64.4 ± 5.2 %, P &lt;0.05). Myocardial phosphocreatine-to-ATP ratio was lower in TAC mice when compared to healthy controls (0.8±0.2 versus 1.2±0.2, P &lt;0.05). Conclusion: Decreased EF in TAC mice is accompanied by decreased phosphocreatine-to-ATP ratio, indicating a disturbed energy homeostasis in this mouse model of heart failure. This research was funded by a VIDI grant from the Netherlands Organisation for Scientific Research (NWO)

Mimicking cardiac fibrosis in a dish:fibroblast density rather than collagen density weakens cardiomyocyte function

\u3cp\u3eCardiac fibrosis is one of the most devastating effects of cardiac disease. Current in vitro models of cardiac fibrosis do not sufficiently mimic the complex in vivo environment of the cardiomyocyte. We determined the local composition and mechanical properties of the myocardium in established mouse models of genetic and acquired fibrosis and tested the effect of myocardial composition on cardiomyocyte contractility in vitro by systematically manipulating the number of fibroblasts and collagen concentration in a platform of engineered cardiac microtissues. The in vitro results showed that while increasing collagen content had little effect on microtissue contraction, increasing fibroblast density caused a significant reduction in contraction force. In addition, the beating frequency dropped significantly in tissues consisting of 50% cardiac fibroblasts or higher. Despite apparent dissimilarities between native and in vitro fibrosis, the latter allows for the independent analysis of local determinants of fibrosis, which is not possible in vivo.\u3c/p\u3

Assessment of myocardial fibrosis in mice using a T2*-weighted 3D radial magnetic resonance imaging sequence

\u3cp\u3eBACKGROUND: Myocardial fibrosis is a common hallmark of many diseases of the heart. Late gadolinium enhanced MRI is a powerful tool to image replacement fibrosis after myocardial infarction (MI). Interstitial fibrosis can be assessed indirectly from an extracellular volume fraction measurement using contrast-enhanced T1 mapping. Detection of short T2* species resulting from fibrotic tissue may provide an attractive non-contrast-enhanced alternative to directly visualize the presence of both replacement and interstitial fibrosis.\u3c/p\u3e\u3cp\u3eOBJECTIVE: To goal of this paper was to explore the use of a T2*-weighted radial sequence for the visualization of fibrosis in mouse heart.\u3c/p\u3e\u3cp\u3eMETHODS: C57BL/6 mice were studied with MI (n = 20, replacement fibrosis), transverse aortic constriction (TAC) (n = 18, diffuse fibrosis), and as control (n = 10). 3D center-out radial T2*-weighted images with varying TE were acquired in vivo and ex vivo (TE = 21 μs-4 ms). Ex vivo T2*-weighted signal decay with TE was analyzed using a 3-component model. Subtraction of short- and long-TE images was used to highlight fibrotic tissue with short T2*. The presence of fibrosis was validated using histology and correlated to MRI findings.\u3c/p\u3e\u3cp\u3eRESULTS: Detailed ex vivo T2*-weighted signal analysis revealed a fast (T2*fast), slow (T2*slow) and lipid (T2*lipid) pool. T2*fast remained essentially constant. Infarct T2*slow decreased significantly, while a moderate decrease was observed in remote tissue in post-MI hearts and in TAC hearts. T2*slow correlated with the presence of diffuse fibrosis in TAC hearts (r = 0.82, P = 0.01). Ex vivo and in vivo subtraction images depicted a positive contrast in the infarct co-localizing with the scar. Infarct volumes from histology and subtraction images linearly correlated (r = 0.94, P&lt;0.001). Region-of-interest analysis in the in vivo post-MI and TAC hearts revealed significant T2* shortening due to fibrosis, in agreement with the ex vivo results. However, in vivo contrast on subtraction images was rather poor, hampering a straightforward visual assessment of the spatial distribution of the fibrotic tissue.\u3c/p\u3