A Multifunctional Nanostructured Hydrogel as a Platform for Deciphering Niche Interactions of Hematopoietic Stem and Progenitor Cells.

For over half a century, hematopoietic stem cells (HSCs) have been used for transplantation therapy to treat severe hematologic diseases. Successful outcomes depend on collecting sufficient donor HSCs as well as ensuring efficient engraftment. These processes are influenced by dynamic interactions of HSCs with the bone marrow niche, which can be revealed by artificial niche models. Here, a multifunctional nanostructured hydrogel is presented as a 2D platform to investigate how the interdependencies of cytokine binding and nanopatterned adhesive ligands influence the behavior of human hematopoietic stem and progenitor cells (HSPCs). The results indicate that the degree of HSPC polarization and motility, observed when cultured on gels presenting the chemokine SDF-1α and a nanoscale-defined density of a cellular (IDSP) or extracellular matrix (LDV) α4ß1 integrin binding motif, are differently influenced on hydrogels functionalized with the different ligand types. Further, SDF-1α promotes cell polarization but not motility. Strikingly, the degree of differentiation correlates negatively with the nanoparticle spacing, which determines ligand density, but only for the cellular-derived IDSP motif. This mechanism potentially offers a means of predictably regulating early HSC fate decisions. Consequently, the innovative multifunctional hydrogel holds promise for deciphering dynamic HSPC-niche interactions and refining transplantation therapy protocols.

Compressed-sensing accelerated 4D flow MRI of cerebrospinal fluid dynamics.

BACKGROUND: 4D flow magnetic resonance imaging (MRI) of CSF can make an important contribution to the understanding of hydrodynamic changes in various neurological diseases but remains limited in clinical application due to long acquisition times. The aim of this study was to evaluate the accuracy of compressed SENSE accelerated MRI measurements of the spinal CSF flow. METHODS: In 20 healthy subjects 4D flow MRI of the CSF in the cervical spine was acquired using compressed sensitivity encoding [CSE, a combination of compressed sensing and parallel imaging (SENSE) provided by the manufacturer] with acceleration factors between 4 and 10. A conventional scan using SENSE was used as reference. Extracted parameters were peak velocity, absolute net flow, forward flow and backward flow. Bland-Altman analysis was performed to determine the scan-rescan reproducibility and the agreement between SENSE and compressed SENSE. Additionally, a time accumulated flow error was calculated. In one additional subject flow of the spinal canal at the level of the entire spinal cord was assessed. RESULTS: Averaged acquisition times were 10:21 min (SENSE), 9:31 min (CSE4), 6:25 min (CSE6), 4:53 min (CSE8) and 3:51 min (CSE10). Acquisition of the CSF flow surrounding the entire spinal cord took 14:40 min. Bland-Altman analysis showed good agreement for peak velocity, but slight overestimations for absolute net flow, forward flow and backward flow (< 1 ml/min) in CSE4-8. Results of the accumulated flow error were similar for CSE4 to CSE8. CONCLUSION: A quantitative analysis of acceleration factors CSE4-10 showed that CSE with an acceleration factor up to 6 is feasible. This allows a scan time reduction of 40% and enables the acquisition and analysis of the CSF flow dynamics surrounding the entire spinal cord within a clinically acceptable scan time.

Adenosine receptors regulate gap junction coupling of the human cerebral microvascular endothelial cells hCMEC/D3 by Ca2+ influx through cyclic nucleotide-gated channels.

KEY POINTS: Gap junction channels are essential for the formation and regulation of physiological units in tissues by allowing the lateral cell-to-cell diffusion of ions, metabolites and second messengers. Stimulation of the adenosine receptor subtype A2B increases the gap junction coupling in the human blood-brain barrier endothelial cell line hCMEC/D3. Although the increased gap junction coupling is cAMP-dependent, neither the protein kinase A nor the exchange protein directly activated by cAMP were involved in this increase. We found that cAMP activates cyclic nucleotide-gated (CNG) channels and thereby induces a Ca2+ influx, which leads to the increase in gap junction coupling. The report identifies CNG channels as a possible physiological link between adenosine receptors and the regulation of gap junction channels in endothelial cells of the blood-brain barrier. ABSTRACT: The human cerebral microvascular endothelial cell line hCMEC/D3 was used to characterize the physiological link between adenosine receptors and the gap junction coupling in endothelial cells of the blood-brain barrier. Expressed adenosine receptor subtypes and connexin (Cx) isoforms were identified by RT-PCR. Scrape loading/dye transfer was used to evaluate the impact of the A2A and A2B adenosine receptor subtype agonist 2-phenylaminoadenosine (2-PAA) on the gap junction coupling. We found that 2-PAA stimulated cAMP synthesis and enhanced gap junction coupling in a concentration-dependent manner. This enhancement was accompanied by an increase in gap junction plaques formed by Cx43. Inhibition of protein kinase A did not affect the 2-PAA-related enhancement of gap junction coupling. In contrast, the cyclic nucleotide-gated (CNG) channel inhibitor l-cis-diltiazem, as well as the chelation of intracellular Ca2+ with BAPTA, or the absence of external Ca2+ , suppressed the 2-PAA-related enhancement of gap junction coupling. Moreover, we observed a 2-PAA-dependent activation of CNG channels by a combination of electrophysiology and pharmacology. In conclusion, the stimulation of adenosine receptors in hCMEC/D3 cells induces a Ca2+ influx by opening CNG channels in a cAMP-dependent manner. Ca2+ in turn induces the formation of new gap junction plaques and a consecutive sustained enhancement of gap junction coupling. The report identifies CNG channels as a physiological link that integrates gap junction coupling into the adenosine receptor-dependent signalling of endothelial cells of the blood-brain barrier.

Data of the molecular dynamics simulations of mutations in the human connexin46 docking interface.

The structure of hCx26 derived from the X-ray analysis was used to generate a homology model for hCx46. Interacting connexin molecules were used as starting model for the molecular dynamics (MD) simulation using NAMD and allowed us to predict the dynamic behavior of hCx46wt and the cataract related mutant hCx46N188T as well as two artificial mutants hCx46N188Q and hCx46N188D. Within the 50 ns simulation time the docked complex composed of the mutants dissociate while hCx46wt remains stable. The data indicates that one hCx46 molecule forms 5-7 hydrogen bonds (HBs) with the counterpart connexin of the opposing connexon. These HBs appear essential for a stable docking of the connexons as shown by the simulation of an entire gap junction channel and were lost for all the tested mutants. The data described here are related to the research article entitled "The cataract related mutation N188T in human connexin46 (hCx46) revealed a critical role for residue N188 in the docking process of gap junction channels" (Schadzek et al., 2015) [1].

Evaluation of incidence rates in pre-clinical studies using a Williams-type procedure.

The analysis of dose-response relationships is a common problem in pre-clinical studies. For example, proportions such as mortality rates and histopathological findings are of particular interest in repeated toxicity studies. Commonly applied designs consist of an untreated control group and several, possibly unequally spaced, dosage groups. The Williams test can be formulated as a multiple contrast test and is a powerful option to evaluate such data. In this paper, we consider simultaneous inference for Williams-type multiple contrasts when the response variable is binomial and sample sizes are only moderate. Approximate simultaneous confidence limits can be constructed using the quantiles of a multivariate normal distribution taking the correlation into account. Alternatively, multiplicity-adjusted p-values can be calculated as well. A simulation study shows that a simple correction based on adding pseudo observations leads to acceptable performance for moderate sample sizes, such as 40 per group. In addition, the calculation of adjusted p-values and approximate power is presented. Finally, the proposed methods are applied to example data from two toxicological studies; the methods are available in an R-package.

Asymptotic simultaneous confidence intervals for many-to-one comparisons of binary proportions in randomized clinical trials.

The simultaneous comparison of proportions of success between many treatments and one control group is a common problem in randomized clinical trials or toxicity studies. In this article, three recently recommended asymptotic confidence interval approaches for the difference of proportions are adjusted for multiplicity, taking the correlation into account. The coverage probability of the resulting interval methods is compared in a simulation study using parameter settings relevant for clinical trials. For moderate to small sample sizes, a method adding two successes and two failures can be recommended. The usage of the proposed methods is illustrated by two examples; an R package is available.

Poly-k-trend tests for survival adjusted analysis of tumor rates formulated as approximate multiple contrast test.

In long-term rodent carcinogenicity studies without cause of death information, poly-k-adjusted tumor rates are commonly used to assess the carcinogenic potential of a compound. Testing trend on proportions, global tests such as the Cochran-Armitage test and Williams-type test have been proposed. Here are introduced simultaneous confidence intervals and adjusted p-values for multiple contrasts on poly-k-adjusted tumor rates, based on approximation with the multivariate normal distribution. Williams-type contrasts and Dunnett-type comparisons to control are special cases of this approach. In simulation studies, the acceptable performance for finite sample sizes is demonstrated. The methods are applied to a real data example and are implemented in a package for R.